Treatment of pain

ABSTRACT

Use of a therapeutic molecule, for the treatment of specific pain conditions, wherein the therapeutic molecule is a single chain, polypeptide fusion protein, comprising: a non-cytotoxic protease, or a fragment thereof, which protease or protease fragment can cleave a protein of the exocytic fusion apparatus of a nociceptive sensory afferent; a Targeting Moiety that can bind to a Binding Site on the nociceptive sensory afferent, which Binding Site can undergo endocytosis to be incorporated into an endosome within the nociceptive sensory afferent; a protease cleavage site at which site the fusion protein is cleavable by a protease, wherein the protease cleavage site is located between the non-cytotoxic protease or fragment thereof and the Targeting Moiety; and a translation domain that can translocate the protease or protease fragment from within an endosome, across the endosomal membrane and into the cytosol of the nociceptive sensory afferent.

This invention relates to the use of non-cytotoxic fusion proteins forthe treatment of specific types of pain.

Toxins may be generally divided into two groups according to the type ofeffect that they have on a target cell. In more detail, the first groupof toxins kill their natural target cells, and are therefore known ascytotoxic toxin molecules. This group of toxins is exemplified interalia by plant toxins such as ricin, and abrin, and by bacterial toxinssuch as diphtheria toxin, and Pseudomonas exotoxin A. Cytotoxic toxinshave attracted much interest in the design of “magic bullets” (e.g.immunoconjugates, which comprise a cytotoxic toxin component and anantibody that binds to a specific marker on a target cell) for thetreatment of cellular disorders and conditions such as cancer. Cytotoxictoxins typically kill their target cells by inhibiting the cellularprocess of protein synthesis.

The second group of toxins, which are known as non-cytotoxic toxins, donot (as their name confirms) kill their natural target cells.Non-cytotoxic toxins have attracted much less commercial interest thanhave their cytotoxic counterparts, and exert their effects on a targetcell by inhibiting cellular processes other than protein synthesis.Non-cytotoxic toxins are produced by a variety of plants, and by avariety of microorganisms such as Clostridium sp. and Neisseria sp.

Clostridial neurotoxins are proteins that typically have a molecularmass of the order of 150 kDa. They are produced by various species ofbacteria, especially of the genus Clostridium, most importantly C.tetani and several strains of C. botulinum, C. butyricum and C.argentinense. There are at present eight different classes of theclostridial neurotoxin, namely: tetanus toxin, and botulinum neurotoxinin its serotypes A, B, C1, D, E, F and G, and they all share similarstructures and modes of action.

Clostridial neurotoxins represent a major group of non-cytotoxic toxinmolecules, and are synthesised by the host bacterium as singlepolypeptides that are modified post-translationally by a proteolyticcleavage event to form two polypeptide chains joined together by adisulphide bond. The two chains are termed the heavy chain (H-chain),which has a molecular mass of approximately 100 kDa, and the light chain(L-chain), which has a molecular mass of approximately 50 kDa.

L-chains possess a protease function (zinc-dependent endopeptidaseactivity) and exhibit a high substrate specificity for vesicle and/orplasma membrane associated proteins involved in the exocytic process.L-chains from different clostridial species or serotypes may hydrolysedifferent but specific peptide bonds in one of three substrate proteins,namely synaptobrevin, syntaxin or SNAP-25. These substrates areimportant components of the neurosecretory machinery.

Neisseria sp., most importantly from the species N. gonorrhoeae, producefunctionally similar non-cytotoxic proteases. An example of such aprotease is IgA protease (see WO99/58571).

It has been well documented in the art that toxin molecules may bere-targeted to a cell that is not the toxin's natural target cell. Whenso re-targeted, the modified toxin is capable of binding to a desiredtarget cell and, following subsequent translocation into the cytosol, iscapable of exerting its effect on the target cell. Said re-targeting isachieved by replacing the natural Targeting Moiety (TM) of the toxinwith a different TM. In this regard, the TM is selected so that it willbind to a desired target cell, and allow subsequent passage of themodified toxin into an endosome within the target cell. The modifiedtoxin also comprises a translocation domain to enable entry of thenon-cytotoxic protease into the cell cytosol. The translocation domaincan be the natural translocation domain of the toxin or it can be adifferent translocation domain obtained from a microbial protein withtranslocation activity.

The above-mentioned TM replacement may be effected by conventionalchemical conjugation techniques, which are well known to a skilledperson. In this regard, reference is made to Hermanson, G. T. (1996),Bioconjugate techniques, Academic Press, and to Wong, S. S. (1991),Chemistry of protein conjugation and cross-linking, CRC Press.

Chemical conjugation is, however, often imprecise. For example,following conjugation, a TM may become joined to the remainder of theconjugate at more than one attachment site.

Chemical conjugation is also difficult to control. For example, a TM maybecome joined to the remainder of the modified toxin at an attachmentsite on the protease component and/or on the translocation component.This is problematic when attachment to only one of said components(preferably at a single site) is desired for therapeutic efficacy.

Thus, chemical conjugation results in a mixed population of modifiedtoxin molecules, which is undesirable.

As an alternative to chemical conjugation, TM replacement may beeffected by recombinant preparation of a single polypeptide fusionprotein (see WO98/07864). This technique is based on the in vivobacterial mechanism by which native clostridial neurotoxin (i.e.holotoxin) is prepared, and results in a fusion protein having thefollowing structural arrangement:

NH₂-[protease component]-[translocation component]-[TM]-COOH

According to WO98/07864, the TM is placed towards the C-terminal end ofthe fusion protein. The fusion protein is then activated by treatmentwith a protease, which cleaves at a site between the protease componentand the translocation component. A di-chain protein is thus produced,comprising the protease component as a single polypeptide chaincovalently attached (via a disulphide bridge) to another singlepolypeptide chain containing the translocation component plus TM. Whilstthe WO98/07864 methodology follows (in terms of structural arrangementof the fusion protein) the natural expression system of clostridialholotoxin, the present inventors have found that this system may resultin the production of certain fusion proteins that have asubstantially-reduced binding ability for the intended target cell.

This problem is particularly relevant in the context of treatingspecific types of pain.

The present invention addresses one or more of the above-mentionedproblems by providing use of a therapeutic molecule for the manufactureof a medicament for the treatment of particular types of pain, whereinthe therapeutic molecule is a single chain, polypeptide fusion protein,comprising:

-   -   a. a non-cytotoxic protease, or a fragment thereof, which        protease or protease fragment is capable of cleaving a protein        of the exocytic fusion apparatus in a nociceptive sensory        afferent;    -   b. a Targeting Moiety that is capable of binding to a Binding        Site on the nociceptive sensory afferent, which Binding Site is        capable of undergoing endocytosis to be incorporated into an        endosome within the nociceptive sensory afferent;    -   c. a protease cleavage site at which site the fusion protein is        cleavable by a protease, wherein the protease cleavage site is        located between the non-cytotoxic protease or fragment thereof        and the Targeting Moiety; and    -   d. a translocation domain that is capable of translocating the        protease or protease fragment from within an endosome, across        the endosomal membrane and into the cytosol of the nociceptive        sensory afferent.

The present inventors have found that the WO 98/07864 fusion proteinsystem is not optimal for TMs requiring a N-terminal domain forinteraction with a binding site on a nociceptive sensory afferent. Thisproblem is particularly acute with TMs that require a specificN-terminus amino acid residue or a specific sequence of amino acidresidues including the N-terminus amino acid residue for interactionwith a binding site on a nociceptive sensory afferent.

In contrast to WO98/07864, the present invention employs non-cytotoxicfusion proteins, wherein the TM component of the fusion includes therelevant binding domain in an intra domain or an amino acid sequencelocated towards the middle (ie. of the linear peptide sequence) of theTM, or preferably located towards the N-terminus of the TM, or morepreferably at or near to the N-terminus. The N-terminal domain iscapable of binding to a Binding Site on a nociceptive sensory afferent,and the TM preferably has a requirement for a specific and definedsequence of amino acid residue(s) to be free at its N-terminus.

The compounds described here may be used to treat a patient sufferingfrom one or more types of chronic pain including neuropathic pain,inflammatory pain, headache pain, somatic pain, visceral pain, andreferred pain.

To “treat,” as used here, means to deal with medically. It includes, forexample, administering a compound of the invention to prevent pain or tolessen its severity.

The term “pain,” as used here, means any unpleasant sensory experience,usually associated with a physical disorder. The physical disorder mayor may not be apparent to a clinician. Pain is of two types: chronic andacute. An “acute pain” is a pain of short duration having a suddenonset. One type of acute pain, for example, is cutaneous pain felt oninjury to the skin or other superficial tissues, such as caused by a cutor a burn. Cutaneous nociceptors terminate just below the skin, and dueto the high concentration of nerve endings, produce a well-defined,localized pain of short duration. “Chronic pain” is a pain other than anacute pain. Chronic pain includes neuropathic pain, inflammatory pain,headache pain, somatic pain visceral pain and referred pain.

I. Neuropathic Pain

The compounds of the invention may be used to treat pain caused by orotherwise associated with any of the following neuropathic painconditions. “Neuropathic pain” means abnormal sensory input, resultingin discomfort, from the peripheral nervous system, central nervoussystems, or both.

A. Symptoms of Neuropathic Pain

Symptoms of neuropathic pain can involve persistent, spontaneous pain,as well as allodynia (a painful response to a stimulus that normally isnot painful), hyperalgesia (an accentuated response to a painfulstimulus that usually causes only a mild discomfort, such as a pinprick), or hyperpathia (where a short discomfort becomes a prolongedsevere pain).

B. Causes of Neuropathic Pain

Neuropathic pain may be caused by any of the following.

1. A traumatic insult, such as, for example, a nerve compression injury(e.g., a nerve crush, a nerve stretch, a nerve entrapment or anincomplete nerve transsection); a spinal cord injury (e.g., ahemisection of the spinal cord); a limb amputation; a contusion; aninflammation (e.g., an inflammation of the spinal cord); or a surgicalprocedure.2. An ischemic event, including, for example, a stroke and heart attack.3. An infectious agent4. Exposure to a toxic agent, including, for example, a drug, analcohol, a heavy metal (e.g., lead, arsenic, mercury), an industrialagent (e.g., a solvent, fumes from a glue) or nitrous oxide.5. A disease, including, for example, an inflammatory disorder, aneoplastic tumor, an acquired immune deficiency syndrome (AIDS), Lymesdisease, a leprosy, a metabolic disease, a peripheral nerve disorder,like neuroma, a mononeuropathy or a polyneuropathy.C. Types of neuropathic pain

1. Neuralgia.

A neuralgia is a pain that radiates along the course of one or morespecific nerves usually without any demonstrable pathological change inthe nerve structure. The causes of neuralgia are varied. Chemicalirritation, inflammation, trauma (including surgery), compression bynearby structures (for instance, tumors), and infections may all lead toneuralgia. In many cases, however, the cause is unknown orunidentifiable. Neuralgia is most common in elderly persons, but it mayoccur at any age. A neuralgia, includes, without limitation, atrigeminal neuralgia, a post-herpetic neuralgia, a postherpeticneuralgia, a glossopharyngeal neuralgia, a sciatica and an atypicalfacial pain.

Neuralgia is pain in the distribution of a nerve or nerves. Examples aretrigeminal neuralgia, atypical facial pain, and postherpetic neuralgia(caused by shingles or herpes). The affected nerves are responsible forsensing touch, temperature and pressure in the facial area from the jawto the forehead. The disorder generally causes short episodes ofexcruciating pain, usually for less than two minutes and on only oneside of the face. The pain can be described in a variety of ways such as“stabbing,” “sharp,” “like lightning,” “burning,” and even “itchy”. Inthe atypical form of TN, the pain can also present as severe or merelyaching and last for extended periods. The pain associated with TN isrecognized as one the most excruciating pains that can be experienced.

Simple stimuli such as eating, talking, washing the face, or any lighttouch or sensation can trigger an attack (even the sensation of a gentlebreeze). The attacks can occur in clusters or as an isolated attack.

Symptoms include sharp, stabbing pain or constant, burning pain locatedanywhere, usually on or near the surface of the body, in the samelocation for each episode; pain along the path of a specific nerve;impaired function of affected body part due to pain, or muscle weaknessdue to concomitant motor nerve damage; increased sensitivity of the skinor numbness of the affected skin area (feeling similar to a localanesthetic such as a Novacaine shot); and any touch or pressure isinterpreted as pain. Movement may also be painful.

Trigeminal neuralgia is the most common form of neuralgia. It affectsthe main sensory nerve of the face, the trigeminal nerve (“trigeminal”literally means “three origins”, referring to the division of the nerveinto 3 branches). This condition involves sudden and short attacks ofsevere pain on the side of the face, along the area supplied by thetrigeminal nerve on that side. The pain attacks may be severe enough tocause a facial grimace, which is classically referred to as a painfultic (tic douloureux). Sometimes, the cause of trigeminal neuralgia is ablood vessel or small tumor pressing on the nerve. Disorders such asmultiple sclerosis (an inflammatory disease affecting the brain andspinal cord), certain forms of arthritis, and diabetes (high bloodsugar) may also cause trigeminal neuralgia, but a cause is not alwaysidentified. In this condition, certain movements such as chewing,talking, swallowing, or touching an area of the face may trigger a spasmof excruciating pain.

A related but rather uncommon neuralgia affects the glosso-pharyngealnerve, which provides sensation to the throat. Symptoms of thisneuralgia are short, shock-like episodes of pain located in the throat.

Neuralgia may occur after infections such as shingles, which is causedby the varicella-zoster virus, a type of herpesvirus. This neuralgiaproduces a constant burning pain after the shingles rash has healed. Thepain is worsened by movement of or contact with the affected area. Notall of those diagnosed with shingles go on to experience postherpeticneuralgia, which can be more painful than shingles. The pain andsensitivity can last for months or even years. The pain is usually inthe form of an intolerable sensitivity to any touch but especially lighttouch. Postherpetic neuralgia is not restricted to the face; it canoccur anywhere on the body but usually occurs at the location of theshingles rash.

Depression is not uncommon due to the pain and social isolation duringthe illness.

Postherpetic neuralgia may be debilitating long after signs of theoriginal herpes infection have disappeared. Other infectious diseasesthat may cause neuralgia are syphilis and Lyme disease.

Diabetes is another common cause of neuralgia. This very common medicalproblem affects almost 1 out of every 20 Americans during adulthood.Diabetes damages the tiny arteries that supply circulation to thenerves, resulting in nerve fiber malfunction and sometimes nerve loss.Diabetes can produce almost any neuralgia, including trigeminalneuralgia, carpal tunnel syndrome (pain and numbness of the hand andwrist), and meralgia paresthetica (numbness and pain in the thigh due todamage to the lateral femoral cutaneous nerve). Strict control of bloodsugar may prevent diabetic nerve damage and may accelerate recovery inpatients who do develop neuralgia.

Other medical conditions that may be associated with neuralgias arechronic renal insufficiency and porphyria—a hereditary disease in whichthe body cannot rid itself of certain substances produced after thenormal breakdown of blood in the body. Certain drugs may also cause thisproblem.

2. Deafferentation.

Deafferentation indicates a loss of the sensory input from a portion ofthe body, and can be caused by interruption of either peripheral sensoryfibres or nerves from the central nervous system. A deafferentation painsyndrome, includes, without limitation, an injury to the brain or spinalcord, a post-stroke pain, a phantom pain, a paraplegia, a brachialplexus avulsion injuries, lumbar radiculopathies.

3. Complex Regional Pain Syndromes (CRPSs)

CRPS is a chronic pain syndrome resulting fromsympathetically-maintained pain, and presents in two forms. CRPS 1currently replaces the term “reflex sympathetic dystrophy syndrome”. Itis a chronic nerve disorder that occurs most often in the arms or legsafter a minor or major injury. CRPS 1 is associated with severe pain;changes in the nails, bone, and skin; and an increased sensitivity totouch in the affected limb. CRPS 2 replaces the term causalgia, andresults from an identified injury to the nerve. A CRPS, includes,without limitation, a CRPS Type I (reflex sympathetic dystrophy) and aCRPS Type II (causalgia).

4. Neuropathy.

A neuropathy is a functional or pathological change in a nerve and ischaracterized clinically by sensory or motor neuron abnormalities.

Central neuropathy is a functional or pathological change in the centralnervous system.

Peripheral neuropathy is a functional or pathological change in one ormore peripheral nerves. The peripheral nerves relay information fromyour central nervous system (brain and spinal cord) to muscles and otherorgans and from your skin, joints, and other organs back to your brain.Peripheral neuropathy occurs when these nerves fail to carry informationto and from the brain and spinal cord, resulting in pain, loss ofsensation, or inability to control muscles. In some cases, the failureof nerves that control blood vessels, intestines, and other organsresults in abnormal blood pressure, digestion problems, and loss ofother basic body processes. Risk factors for neuropathy includediabetes, heavy alcohol use, and exposure to certain chemicals anddrugs. Some people have a hereditary predisposition for neuropathy.Prolonged pressure on a nerve is another risk for developing a nerveinjury. Pressure injury may be caused by prolonged immobility (such as along surgical procedure or lengthy illness) or compression of a nerve bycasts, splints, braces, crutches, or other devices. Polyneuropathyimplies a widespread process that usually affects both sides of the bodyequally. The symptoms depend on which type of nerve is affected. Thethree main types of nerves are sensory, motor, and autonomic. Neuropathycan affect any one or a combination of all three types of nerves.Symptoms also depend on whether the condition affects the whole body orjust one nerve (as from an injury). The cause of chronic inflammatorypolyneuropathy is an abnormal immune response. The specific antigens,immune processes, and triggering factors are variable and in many casesare unknown. It may occur in association with other conditions such asHIV, inflammatory bowel disease, lupus erythematosis, chronic activehepatitis, and blood cell abnormalities.

Peripheral neuropathy may involve a function or pathological change to asingle nerve or nerve group (monneuropathy) or a function orpathological change affecting multiple nerves (polyneuropathy).

Peripheral neuropathiesHereditary disorders

-   -   Charcot-Marie-Tooth disease    -   Friedreich's ataxia        Systemic or metabolic disorders    -   Diabetes (diabetic neuropathy)    -   Dietary deficiencies (especially vitamin B-12)    -   Excessive alcohol use (alcoholic neuropathy)    -   Uremia (from kidney failure)    -   Cancer        Infectious or inflammatory conditions    -   AIDS    -   Hepatitis    -   Colorado tick fever    -   diphtheria    -   Guillain-Barre syndrome    -   HIV infection without development of AIDS    -   leprosy    -   Lyme    -   polyarteritis nodosa    -   rheumatoid arthritis    -   sarcoidosis    -   Sjogren syndrome    -   syphilis    -   systemic lupus erythematosus    -   amyloid        Exposure to toxic compounds    -   sniffing glue or other toxic compounds    -   nitrous oxide    -   industrial agents—especially solvents    -   heavy metals (lead, arsenic, mercury, etc.)    -   Neuropathy secondary to drugs like analgesic nephropathy        Miscellaneous causes    -   ischemia (decreased oxygen/decreased blood flow)    -   prolonged exposure to cold temperature

a. Polyneuropathy

Polyneuropathy is a peripheral neuropathy involving the loss of movementor sensation to an area caused by damage or destruction to multipleperipheral nerves. Polyneuropathic pain, includes, without limitation,post-polio syndrome, postmastectomy syndrome, diabetic neuropathy,alcohol neuropathy, amyloid, toxins, AIDS, hypothyroidism, uremia,vitamin deficiencies, chemotherapy-induced pain, 2′,3′-didexoycytidine(ddC) treatment, Guillain-Barré syndrome or Fabry's disease.

b. Mononeuropathy

Mononeuropathy is a peripheral neuropathy involving loss of movement orsensation to an area caused by damage or destruction to a singleperipheral nerve or nerve group. Mononeuropathy is most often caused bydamage to a local area resulting from injury or trauma, althoughoccasionally systemic disorders may cause isolated nerve damage (as withmononeuritis multiplex). The usual causes are direct trauma, prolongedpressure on the nerve, and compression of the nerve by swelling orinjury to nearby body structures. The damage includes destruction of themyelin sheath (covering) of the nerve or of part of the nerve cell (theaxon). This damage slows or prevents conduction of impulses through thenerve. Mononeuropathy may involve any part of the body. Mononeuropathicpain, includes, without limitation, a sciatic nerve dysfunction, acommon peroneal nerve dysfunction. a radial nerve dysfunction, an ulnarnerve dysfunction, a cranial mononeuropathy VI, a cranial mononeuropathyVII, a cranial mononeuropathy III (compression type), a cranialmononeuropathy III (diabetic type), an axillary nerve dysfunction, acarpal tunnel syndrome, a femoral nerve dysfunction, a tibial nervedysfunction, a Bell's palsy, a thoracic outlet syndrome, a carpal tunnelsyndrome and a sixth (abducent) nerve palsy

c. Generalized Peripheral Neuropathies

Generalized peripheral neuropathis are symmetrical, and usually due tovarious systematic illnesses and disease processes that affect theperipheral nervous system in its entirety. They are further subdividedinto several categories:

i. Distal axonopathies are the result of some metabolic or toxicderangement of neurons. They may be caused by metabolic diseases such asdiabetes, renal failure, deficiency syndromes such as malnutrition andalcoholism, or the effects of toxins or drugs. Distal axonopathy (akadying back neuropathy) is a type of peripheral neuropathy that resultsfrom some metabolic or toxic derangement of peripheral nervous system(PNS) neurons. It is the most common response of nerves to metabolic ortoxic disturbances, and as such may be caused by metabolic diseases suchas diabetes, renal failure, deficiency syndromes such as malnutritionand alcoholism, or the effects of toxins or drugs. The most common causeof distal axonopathy is diabetes, and the most common distal axonopathyis diabetic neuropathy.

ii. Myelinopathies are due to a primary attack on myelin causing anacute failure of impulse conduction. The most common cause is acuteinflammatory demyelinating polyneuropathy (AIDP; aka Guillain-Barrésyndrome), though other causes include chronic inflammatorydemyelinating syndrome (CIDP), genetic metabolic disorders (e.g.,leukodystrophy), or toxins. Myelinopathy is due to primary destructionof myelin or the myelinating Schwann cells, which leaves the axonintact, but causes an acute failure of impulse conduction. Thisdemyelination slows down or completely blocks the conduction ofelectical impulses through the nerve. The most common cause is acuteinflammatory demyelinating polyneuropathy (AIDP, better known asGuillain-Barré syndrome), though other causes include chronicinflammatory demyelinating polyneuropathy (CIDP), genetic metabolicdisorders (e.g., leukodystrophy or Charcot-Marie-Tooth disease), ortoxins.

iii. Neuronopathies are the result of destruction of peripheral nervoussystem (PNS) neurons. They may be caused by motor neurone diseases,sensory neuronopathies (e.g., Herpes zoster), toxins or autonomicdysfunction. Neurotoxins may cause neuronopathies, such as thechemotherapy agent vincristine. Neuronopathy is dysfunction due todamage to neurons of the peripheral nervous system (PNS), resulting in aperipheral neuropathy. It may be caused by motor neurone diseases,sensory neuronopathies (e.g., Herpes zoster), toxic substances orautonomic dysfunction. A person with neuronopathy may present indifferent ways, depending on the cause, the way it affects the nervecells, and the type of nerve cell that is most affected.

iv. Focal entrapment neuropathies (e.g., carpal tunnel syndrome).

II. Inflammatory Pain

The compounds of the invention may be used to treat pain caused by orotherwise associated with any of the following inflammatory conditions

A. Arthritic Disorder

Arthritic disorders include, for example, a rheumatoid arthritis; ajuvenile rheumatoid arthritis; a systemic lupus erythematosus (SLE); agouty arthritis; a scleroderma; an osteoarthritis; a psoriaticarthritis; an ankylosing spondylitis; a Reiter's syndrome (reactivearthritis); an adult Still's disease; an arthritis from a viralinfection; an arthritis from a bacterial infection, such as, e.g., agonococcal arthritis and a non-gonococcal bacterial arthritis (septicarthritis); a Tertiary Lyme disease; a tuberculous arthritis; and anarthritis from a fungal infection, such as, e.g. a blastomycosis

B. Autoimmune Diseases

Autoimmune diseases include, for example, a Guillain-Barré syndrome, aHashimoto's thyroiditis, a pernicious anemia, an Addison's disease, atype I diabetes, a systemic lupus erythematosus, a dermatomyositis, aSjogren's syndrome, a lupus erythematosus, a multiple sclerosis, amyasthenia gravis, a Reiter's syndrome and a Grave's disease.

C. Connective Tissue Disorder

Connective tissue disorders include, for example, a spondyloarthritis adermatomyositis, and a fibromyalgia.

D. Injury

Inflammation caused by injury, including, for example, a crush,puncture, stretch of a tissue or joint, may cause chronic inflammatorypain.

E. Infection

Inflammation caused by infection, including, for example, a tuberculosisor an interstitial keratitis may cause chronic inflammatory pain.

F. Neuritis

Neuritis is an inflammatory process affecting a nerve or group ofnerves. Symptoms depend on the nerves involved, but may include pain,paresthesias, paresis, or hypesthesia (numbness).

Examples Include

a. Brachial neuritis

b. Retrobulbar neuropathy, an inflammatory process affecting the part ofthe optic nerve lying immediately behind the eyeball.

c. Optic neuropathy, an inflammatory process affecting the optic nervecausing sudden, reduced vision in the affected eye. The cause of opticneuritis is unknown. The sudden inflammation of the optic nerve (thenerve connecting the eye and the brain) leads to swelling anddestruction of the myelin sheath. The inflammation may occasionally bethe result of a viral infection, or it may be caused by autoimmunediseases such as multiple sclerosis. Risk factors are related to thepossible causes.

d. Vestibular neuritis, a viral infection causing an inflammatoryprocess affecting the vestibular nerve.

G. Joint Inflammation

Inflammation of the joint, such as that caused by bursitis ortendonitis, for example, may cause chronic inflammatory pain.

III. Headache Pain

The compounds of the invention may be used to treat pain caused by orotherwise associated with any of the following headache conditions. Aheadache (medically known as cephalgia) is a condition of mild to severepain in the head; sometimes neck or upper back pain may also beinterpreted as a headache. It may indicate an underlying local orsystemic disease or be a disorder in itself.

A. Muscular/Myogenic Headache

Muscular/myogenic headaches appear to involve the tightening or tensingof facial and neck muscles; they may radiate to the forehead. Tensionheadache is the most common form of myogenic headache.

A tension headache is a condition involving pain or discomfort in thehead, scalp, or neck, usually associated with muscle tightness in theseareas. Tension headaches result from the contraction of neck and scalpmuscles. One cause of this muscle contraction is a response to stress,depression or anxiety. Any activity that causes the head to be held inone position for a long time without moving can cause a headache. Suchactivities include typing or use of computers, fine work with the hands,and use of a microscope. Sleeping in a cold room or sleeping with theneck in an abnormal position may also trigger this type of headache. Atension-type headache, includes, without limitation, an episodic tensionheadache and a chronic tension headache.

B. Vascular Headache

The most common type of vascular headache is migraine. Other kinds ofvascular headaches include cluster headaches, which cause repeatedepisodes of intense pain, and headaches resulting from high bloodpressure

1. Migraine

A migraine is a heterogeneous disorder that generally involves recurringheadaches. Migraines are different from other headaches because theyoccur with other symptoms, such as, e.g., nausea, vomiting, orsensitivity to light. In most people, a throbbing pain is felt only onone side of the head. Clinical features such as type of aura symptoms,presence of prodromes, or associated symptoms such as vertigo, may beseen in subgroups of patients with different underlyingpathophysiological and genetic mechanisms. A migraine headache,includes, without limitation, a migraine without aura (common migraine),a migraine with aura (classic migraine), a menstrual migraine, amigraine equivalent (acephalic headache), a complicated migraine, anabdominal migraine and a mixed tension migraine.

2. Cluster Headache

Cluster headaches affect one side of the head (unilateral) and may beassociated with tearing of the eyes and nasal congestion. They occurs inclusters, happening repeatedly every day at the same time for severalweeks and then remitting.

D. High Blood Pressure Headache E. Traction and Inflammatory Headache

Traction and inflammatory headaches are usually symptoms of otherdisorders, ranging from stroke to sinus infection.

F. Hormone Headache G. Rebound Headache

Rebound headaches, also known as medication overuse headaches, occurwhen medication is taken too frequently to relieve headache. Reboundheadaches frequently occur daily and can be very painful.

H. Chronic Sinusitis Headache

Sinusitis is inflammation, either bacterial, fungal, viral, allergic orautoimmune, of the paranasal sinuses. Chronic sinusitis is one of themost common complications of the common cold. Symptoms include: Nasalcongestion; facial pain; headache; fever; general malaise; thick greenor yellow discharge; feeling of facial ‘fullness’ worsening on bendingover. In a small number of cases, chronic maxillary sinusitis can alsobe brought on by the spreading of bacteria from a dental infection.Chronic hyperplastic eosinophilic sinusitis is a noninfective form ofchronic sinusitis.

I. An Organic Headache J. Ictal Headaches

Ital headaches are headaches associated with seizure activity.

IV. Somatic Pain

The compounds of the invention may be used to treat pain caused by orotherwise associated with any of the following somatic pain conditions.Somatic pain originates from ligaments, tendons, bones, blood vessels,and even nerves themselves. It is detected with somatic nociceptors. Thescarcity of pain receptors in these areas produces a dull,poorly-localized pain of longer duration than cutaneous pain; examplesinclude sprains and broken bones. Additional examples include thefollowing.

A. Excessive Muscle Tension

Excessive muclse tension can be caused, for example, by a sprain or astrain.

B. Repetitive Motion Disorders

Repetitive motion disorders can result from overuse of the hands,wrists, elbows, shoulders, neck, back, hips, knees, feet, legs, orankles.

C. Muscle Disorders

Muscle disorders causing somatic pain include, for example, apolymyositis, a dermatomyositis, a lupus, a fibromyalgia, a polymyalgiarheumatica, and a rhabdomyolysis.

D. Myalgia

Myalgia is muscle pain and is a symptom of many diseases and disorders.The most common cause for myalgia is either overuse or over-stretchingof a muscle or group of muscles. Myalgia without a traumatic history isoften due to viral infections. Longer-term myalgias may be indicative ofa metabolic myopathy, some nutritional deficiencies or chronic fatiguesyndrome.

E. Infection

Infection can cause somatic pain. Examples of such infection include,for example, an abscess in the muscle, a trichinosis, an influenza, aLyme disease, a malaria, a Rocky Mountain spotted fever, Avianinfluenza, the common cold, community-acquired pneumonia, meningitis,monkeypox, Severe Acute Respiratory Syndrome, toxic shock syndrome,trichinosis, typhoid fever, and upper respiratory tract infection.

F. Drugs

Drugs can cause somatic pain. Such drugs include, for example, cocaine,a statin for lowering cholesterol (such as atorvastatin, simvastatin,and lovastatin), and an ACE inhibitor for lowering blood pressure (suchas enalapril and captopril)

V. Visceral Pain

The compounds of the invention may be used to treat pain caused by orotherwise associated with any of the following visceral pain conditions.Visceral pain originates from body's viscera, or organs. Visceralnociceptors are located within body organs and internal cavities. Theeven greater scarcity of nociceptors in these areas produces pain thatis usually more aching and of a longer duration than somatic pain.Visceral pain is extremely difficult to localise, and several injuriesto visceral tissue exhibit “referred” pain, where the sensation islocalised to an area completely unrelated to the site of injury.Examples of visceral pain include the following.

A. Functional Visceral Pain

Functional visceral pain includes, for example, an irritable bowelsyndrome and a chronic functional abdominal pain (CFAP), a functionalconstipation and a functional dyspepsia, a non-cardiac chest pain (NCCP)and a chronic abdominal pain.

B. Chronic Gastrointestinal Inflammation

Chronic gastrointestinal inflammation includes, for example, agastritis, an inflammatory bowel disease, like, e.g., a Crohn's disease,an ulcerative colitis, a microscopic colitis, a diverticulitis and agastroenteritis; an interstitial cystitis; an intestinal ischemia; acholecystitis; an appendicitis; a gastroesophageal reflux; an ulcer, anephrolithiasis, an urinary tract infection, a pancreatitis and ahernia.

C. Autoimmune Pain

Autoimmune pain includes, for example, a sarcoidosis and a vasculitis.

D. Orangic Visceral Pain

Organic visceral pain includes, for example, pain resulting from atraumatic, inflammatory or degenerative lesion of the gut or produced bya tumor impinging on sensory innervation.

E. Treatment-Induced Visceral Pain

Treatment-induced visceral pain includes, for example, a pain attendantto chemotherapy therapy or a pain attendant to radiation therapy.

VI. Referred Pain

The compounds of the invention may be used to treat pain caused by orotherwise associated with any of the following referred pain conditions.

Referred pain arises from pain localized to an area separate from thesite of pain stimulation. Often, referred pain arises when a nerve iscompressed or damaged at or near its origin. In this circumstance, thesensation of pain will generally be felt in the territory that the nerveserves, even though the damage originates elsewhere. A common exampleoccurs in intervertebral disc herniation, in which a nerve root arisingfrom the spinal cord is compressed by adjacent disc material. Althoughpain may arise from the damaged disc itself, pain will also be felt inthe region served by the compressed nerve (for example, the thigh, knee,or foot). Relieving the pressure on the nerve root may ameliorate thereferred pain, provided that permanent nerve damage has not occurred.Myocardial ischaemia (the loss of blood flow to a part of the heartmuscle tissue) is possibly the best known example of referred pain; thesensation can occur in the upper chest as a restricted feeling, or as anache in the left shoulder, arm or even hand.

The non-cytotoxic protease component of the present invention is anon-cytotoxic protease, or a fragment thereof, which protease orprotease fragment is capable of cleaving different but specific peptidebonds in one of three substrate proteins, namely synaptobrevin, syntaxinor SNAP-25, of the exocytic fusion apparatus in a nociceptive sensoryafferent. These substrates are important components of theneurosecretory machinery. The non-cytotoxic protease component of thepresent invention is preferably a neisserial IgA protease or a fragmentthereof or a clostridial neurotoxin L-chain or a fragment thereof. Aparticularly preferred non-cytotoxic protease component is a botulinumneurotoxin (BoNT) L-chain or a fragment thereof.

The translocation component of the present invention enablestranslocation of the non-cytotoxic protease (or fragment thereof) intothe target cell such that functional expression of protease activityoccurs within the cytosol of the target cell. The translocationcomponent is preferably capable of forming ion-permeable pores in lipidmembranes under conditions of low pH. Preferably it has been found touse only those portions of the protein molecule capable ofpore-formation within the endosomal membrane. The translocationcomponent may be obtained from a microbial protein source, in particularfrom a bacterial or viral protein source. Hence, in one embodiment, thetranslocation component is a translocating domain of an enzyme, such asa bacterial toxin or viral protein. The translocation component of thepresent invention is preferably a clostridial neurotoxin H-chain or afragment thereof. Most preferably it is the H_(N) domain (or afunctional component thereof), wherein H_(N) means a portion or fragmentof the H-chain of a clostridial neurotoxin approximately equivalent tothe amino-terminal half of the H-chain, or the domain corresponding tothat fragment in the intact H-chain.

The TM component of the present invention is responsible for binding thefusion protein of the present invention to a Binding Site on a targetcell. Thus, the TM component is simply a ligand through which a fusionprotein of the present invention binds to a selected target cell.

In the context of the present invention, the target cell is anociceptive sensory afferent, preferably a primary nociceptive afferent(e.g. an A-fibre such as an Aδ-fibre or a C-fibre). Thus, the fusionproteins of the present invention are capable of inhibitingneurotransmitter or neuromodulator [e.g. glutamate, substance P,calcitonin-gene related peptide (CGRP), and/or neuropeptide Y] releasefrom discrete populations of nociceptive sensory afferent neurons. Inuse, the fusion proteins reduce or prevent the transmission of sensoryafferent signals (e.g. neurotransmitters or neuromodulators) fromperipheral to central pain fibres, and therefore have application astherapeutic molecules for the treatment of pain, in particular chronicpain.

It is routine to confirm that a TM binds to a nociceptive sensoryafferent. For example, a simple radioactive displacement experiment maybe employed in which, tissue or cells representative of the nociceptivesensory afferent (for example DRGs) are exposed to labelled (e.g.tritiated) ligand in the presence of an excess of unlabelled ligand. Insuch an experiment, the relative proportions of non-specific andspecific binding may be assessed, thereby allowing confirmation that theligand binds to the nociceptive sensory afferent target cell.Optionally, the assay may include one or more binding antagonists, andthe assay may further comprise observing a loss of ligand binding.Examples of this type of experiment can be found in Hulme, E. C. (1990),Receptor-binding studies, a brief outline, pp. 303-311, In Receptorbiochemistry, A Practical Approach, Ed. E. C. Hulme, Oxford UniversityPress.

The fusion proteins of the present invention generally demonstrate areduced binding affinity (in the region of up to 100-fold) fornociceptive sensory afferent target cells when compared with thecorresponding ‘free’ TM. However, despite this observation, the fusionproteins of the present invention surprisingly demonstrate goodefficacy. This can be attributed to two principal features. First, thenon-cytotoxic protease component is catalytic—thus, the therapeuticeffect of a few such molecules is rapidly amplified. Secondly, thereceptors present on the nociceptive sensory afferents need only act asa gateway for entry of the therapeutic, and need not necessarily bestimulated to a level required in order to achieve a ligand-receptormediated pharmacological response. Accordingly, the fusion proteins ofthe present invention may be administered at a dosage that is much lowerthat would be employed for other types of analgesic molecules such asNSAIDS, morphine, and gabapentin. The latter molecules are typicallyadministered at high microgram to milligram (even up to hundreds ofmilligram) quantities, whereas the fusion proteins of the presentinvention may be administered at much lower dosages, typically at least10-fold lower, and more typically at 100-fold lower.

The TM preferably comprises a maximum of 50 amino acid residues, morepreferably a maximum of 40 amino acid residues, particularly preferablya maximum of 30 amino acid residues, and most preferably a maximum of 20amino acid residues.

Opioids represent a preferred group of TMs of the present invention.Within this family of peptides is included enkephalins (met and leu),endomorphins 1 and 2, β-endorphin and dynorphin. Opioid peptides arefrequently used in the clinic to modify the activity to nociceptors, andother cells involved in the pain response. As exemplified by thethree-step World Health Organisation Analgesic Ladder, opioids haveentry points into the pharmacological treatment of chronic cancer andnon-cancer pain at all three stages, underlining their importance to thetreatment of pain. Reference to opioids embraces fragments, variants andderivatives thereof, which retain the ability to bind to nociceptivesensory afferents.

The TM of the invention can also be a molecule that acts as an “agonist”at one or more of the receptors present on a nociceptive sensoryafferent, more particularly on a primary nociceptive afferent.Conventionally, an agonist has been considered any molecule that caneither increase or decrease activities within a cell, namely anymolecule that simply causes an alteration of cell activity. For example,the conventional meaning of an agonist would include a chemicalsubstance capable of combining with a receptor on a cell and initiatinga reaction or activity, or a drug that induces an active response byactivating receptors, whether the response is an increase or decrease incellular activity.

However, for the purposes of this invention, an agonist is morespecifically defined as a molecule that is capable of stimulating theprocess of exocytic fusion in a target cell, which process issusceptible to inhibition by a protease (or fragment thereof) capable ofcleaving a protein of the exocytic fusion apparatus in said target cell.

Accordingly, the particular agonist definition of the present inventionwould exclude many molecules that would be conventionally considered asagonists. For example, nerve growth factor (NGF) is an agonist inrespect of its ability to promote neuronal differentiation via bindingto a TrkA receptor. However, NGF is not an agonist when assessed by theabove criteria because it is not a principal inducer of exocytic fusion.In addition, the process that NGF stimulates (i.e. cell differentiation)is not susceptible to inhibition by the protease activity of anon-cytotoxic toxin molecule.

The agonist properties of a TM that binds to a receptor on a nociceptiveafferent can be confirmed using the methods described in Example 10.

In a preferred embodiment of the invention, the target for the TM is theORL₁ receptor. This receptor is a member of the G-protein-coupled classof receptors, and has a seven transmembrane domain structure. Theproperties of the ORL₁ receptor are discussed in detail in Mogil &Pasternak (2001), Pharmacological Reviews, Vol. 53, No. 3, pages381-415.

In one embodiment, the TM is a molecule that binds (preferably thatspecifically binds) to the ORL₁ receptor. More preferably, the TM is an“agonist” of the ORL₁ receptor. The term “agonist” in this context isdefined as above.

The agonist properties of a TM that binds to an ORL₁ receptor can beconfirmed using the methods described in Example 10. These methods arebased on previous experiments [see Inoue et al. 1998 [Proc. Natl. Acad.Sci., 95, 10949-10953]), which confirm that the natural agonist of theORL₁ receptor, nociceptin, causes the induction of substance P releasefrom nociceptive primary afferent neurons. This is supported by the factthat:

-   -   the nociceptin-induced responses are abolished by specific NK1        receptor (the substance P receptor) antagonists; and    -   pre-treatment of the cells with capsaicin (which depletes        substance P from small diameter primary afferent neurons)        attenuates the nociceptin-induced responses.

Similarly, Inoue et al. confirm that an intraplantar injection ofbotulinum neurotoxin type A abolishes the nociceptin-induced responses.Since it is known that BoNT inhibits the release of substance P fromprimary afferent neurons (Welch et al., 2000, Toxicon, 38, 245-258),this confirms the link between nociceptin-ORL₁ interaction andsubsequent release of substance P.

Thus, a TM can be said to have agonist activity at the ORL₁ receptor ifthe TM causes an induction in the release of substance P from anociceptive sensory afferent neuron (see Example 10).

In a particularly preferred embodiment of the invention, the TM isnociceptin—the natural ligand for the ORL₁ receptor. Nociceptin targetsthe ORL₁ receptor with high affinity. Examples of other preferred TMsinclude:

Code Sequence Ref. SEQ ID No. Nociceptin 1-17 FGGFTGARKSARKLANQ [1] 37,38 Nociceptin 1-11 FGGFTGARKSA [1] 39, 40 Nociceptin [Y10] 1-11FGGFTGARKYA [1] 41, 42 Nociceptin [Y11] 1-11 FGGFTGARKSY [1] 43, 44Nociceptin [Y14] 1-17 FGGFTGARKSARKYANQ [1] 45, 46 Nociceptin 1-13FGGFTGARKSARK [2] 47, 48 Nociceptin [R14K15] FGGFTGARKSARKRKNQ [3, 4]49, 50 1-17 (also known in this specification as “variant” nociceptin)Peptide agonist Peptide agonists from [5] — combinatorial libraryapproach [1] Mogil & Pasternak, 2001, Pharmacol. Rev., 53, 381-415[2] Maile et al., 2003, Neurosci. Lett., 350, 190-192 [3] Rizzi et al.,2002, J. Pharmacol. Exp. Therap., 300, 57-63 [4] Okada et al., 2000,Biochem. Biophys. Res. Commun., 278, 493-498 [5] Dooley et al., 1997, JPharmacol Exp Ther. 283(2), 735-41.

The above-identified “variant” TM demonstrates particularly good bindingaffinity (when compared with natural nociceptin) for nociceptive sensoryafferents. This is surprising as the amino acid modifications occur at aposition away from the N-terminus of the TM. Moreover, the modificationsare almost at the C-terminus of the TM, which in turn is attached to alarge polypeptide sequence (i.e. the translocation domain). Generallyspeaking, a TM-containing fusion protein will demonstrate an approximate100-fold reduction in binding ability vis-à-vis the TM per se. Theabove-mentioned “variant” TM per se demonstrates an approximate 3- to10-fold increase in binding ability for a nociceptive sensory afferent(e.g. via the ORL1 receptor) vis-à-vis natural nociceptin. Thus, a“variant” TM-containing fusion might be expected to demonstrate anapproximate 10-fold reduction in binding ability for a nociceptivesensory afferent (e.g. via the ORL1 receptor) vis-à-vis ‘free’nociceptin. However, the present inventors have demonstrated that such“variant” TM-containing fusion proteins demonstrate a binding abilitythat (most surprisingly) closely mirrors that of ‘free’ nociceptin—seeFIG. 14.

In the context of the present invention, the term opioid or an agonistof the ORL₁ receptor (such as nociceptin, or any one of the peptideslisted in the table above) embraces molecules having at least 70%,preferably at least 80%, more preferably at least 90%, and mostpreferably at least 95% homology with said opioid or agonist. Theagonist homologues retain the agonist properties of nociceptin at theORL₁ receptor, which may be tested using the methods provided in Example10. Similarly, an opioid homologue substantially retains the bindingfunction of the opioid with which it shows high homology.

The invention also encompasses fragments, variants, and derivatives ofany one of the TMs described above. These fragments, variants, andderivatives substantially retain the properties that are ascribed tosaid TMs.

In addition to the above-mentioned opioid and non-opioid classes of TMs,a variety of other polypeptides are suitable for targeting the fusionproteins of the present invention to nociceptive sensory afferents (e.g.to nociceptors). In this regard, particular reference is made to galaninand derivatives of galanin. Galanin receptors are found pre- andpost-synaptically in DRGs (Liu & Hokfelt, (2002), Trends Pharm. Sci.,23(10), 468-74), and are enhanced in expression during neuropathic painstates. Proteinase-activated receptors (PARs) are also a preferred groupof TMs of the present invention, most particularly PAR-2. It is knownthat agonists of PAR-2 induce/elicit acute inflammation, in part via aneurogenic mechanism. PAR2 is expressed by primary spinal afferentneurons, and PAR2 agonists stimulate release of substance P (SP) andcalcitonin gene-related peptide (CGRP) in peripheral tissues

A particularly preferred set of TMs of the present invention includes:

Ligand Reference Nociceptin Guerrini, et al., (1997) J. Med. Chem., 40,pp. 1789-1793 β-endorphin Blanc, et al., (1983) J. Biol. Chem., 258(13),pp. 8277-8284 Endomorphin-1; Zadina, et al., (1997). Nature, 386, pp.Endomorphin-2 499-502 Dynorphin Fields & Basbaum (2002) Chapter 11, InThe Textbook of Pain, Wall & Melzack eds. Met-enkephalin Fields &Basbaum (2002) Chapter 11, In The Textbook of Pain, Wall & Melzack eds.Leu-enkephalin Fields & Basbaum (2002) Chapter 11, In The Textbook ofPain, Wall & Melzack eds. Galanin Xu et al., (2000) Neuropeptides, 34(3&4), 137-147 PAR-2 peptide Vergnolle et al., (2001) Nat. Med., 7(7),821-826

The protease cleavage site of the present invention allows cleavage(preferably controlled cleavage) of the fusion protein at a positionbetween the non-cytotoxic protease component and the TM component. It isthis cleavage reaction that converts the fusion protein from a singlechain polypeptide into a disulphide-linked, di-chain polypeptide.

According to a preferred embodiment of the present invention, the TMbinds via a domain or amino acid sequence that is located away from theC-terminus of the TM. For example, the relevant binding domain mayinclude an intra domain or an amino acid sequence located towards themiddle (i.e. of the linear peptide sequence) of the TM. Preferably, therelevant binding domain is located towards the N-terminus of the TM,more preferably at or near to the N-terminus.

In one embodiment, the single chain polypeptide fusion may include morethan one proteolytic cleavage site. However, where two or more suchsites exist, they are different, thereby substantially preventing theoccurrence of multiple cleavage events in the presence of a singleprotease. In another embodiment, it is preferred that the single chainpolypeptide fusion has a single protease cleavage site.

The protease cleavage sequence(s) may be introduced (and/or any inherentcleavage sequence removed) at the DNA level by conventional means, suchas by site-directed mutagenesis. Screening to confirm the presence ofcleavage sequences may be performed manually or with the assistance ofcomputer software (e.g. the MapDraw program by DNASTAR, Inc.).

Whilst any protease cleavage site may be employed, the following arepreferred:

Enterokinase (DDDDK↓) Factor Xa (IEGR↓/IDGR↓) TEV (Tobacco Etch virus)(ENLYFQ↓G) Thrombin (LVPR↓GS) PreScission (LEVLFQ↓GP).

Also embraced by the term protease cleavage site is an intein, which isa self-cleaving sequence. The self-splicing reaction is controllable,for example by varying the concentration of reducing agent present.

In use, the protease cleavage site is cleaved and the N-terminal region(preferably the N-terminus) of the TM becomes exposed. The resultingpolypeptide has a TM with an N-terminal domain or an intra domain thatis substantially free from the remainder of the fusion protein. Thisarrangement ensures that the N-terminal component (or intra domain) ofthe TM may interact directly with a Binding Site on a target cell.

In a preferred embodiment, the TM and the protease cleavage site aredistanced apart in the fusion protein by at most 10 amino acid residues,more preferably by at most 5 amino acid residues, and most preferably byzero amino acid residues. Thus, following cleavage of the proteasecleavage site, a fusion is provided with a TM that has an N-terminaldomain that is substantially free from the remainder of the fusion. Thisarrangement ensures that the N-terminal component of the TargetingMoiety may interact directly with a Binding Site on a target cell.

One advantage associated with the above-mentioned activation step isthat the TM only becomes susceptible to N-terminal degradation onceproteolytic cleavage of the fusion protein has occurred. In addition,the selection of a specific protease cleavage site permits selectiveactivation of the polypeptide fusion into a di-chain conformation.

Construction of the single-chain polypeptide fusion of the presentinvention places the protease cleavage site between the TM and thenon-cytotoxic protease component.

It is preferred that, in the single-chain fusion, the TM is locatedbetween the protease cleavage site and the translocation component. Thisensures that the TM is attached to the translocation domain (i.e. asoccurs with native clostridial holotoxin), though in the case of thepresent invention the order of the two components is reversed vis-à-visnative holotoxin. A further advantage with this arrangement is that theTM is located in an exposed loop region of the fusion protein, which hasminimal structural effects on the conformation of the fusion protein. Inthis regard, said loop is variously referred to as the linker, theactivation loop, the inter-domain linker, or just the surface exposedloop (Schiavo et al 2000, Phys. Rev., 80, 717-766; Turton et al., 2002,Trends Biochem. Sci., 27, 552-558).

In one embodiment, in the single chain polypeptide, the non-cytotoxicprotease component and the translocation component are linked togetherby a disulphide bond. Thus, following cleavage of the protease cleavagesite, the polypeptide assumes a di-chain conformation, wherein theprotease and translocation components remain linked together by thedisulphide bond. To this end, it is preferred that the protease andtranslocation components are distanced apart from one another in thesingle chain fusion protein by a maximum of 100 amino acid residues,more preferably a maximum of 80 amino acid residues, particularlypreferably by a maximum of 60 amino acid residues, and most preferablyby a maximum of 50 amino acid residues.

In one embodiment, the non-cytotoxic protease component forms adisulphide bond with the translocation component of the fusion protein.For example, the amino acid residue of the protease component that formsthe disulphide bond is located within the last 20, preferably within thelast 10 C-terminal amino acid residues of the protease component.Similarly, the amino acid residue within the translocation componentthat forms the second part of the disulphide bond may be located withinthe first 20, preferably within the first 10 N-terminal amino acidresidues of the translocation component.

Alternatively, in the single chain polypeptide, the non-cytotoxicprotease component and the TM may be linked together by a disulphidebond. In this regard, the amino acid residue of the TM that forms thedisulphide bond is preferably located away from the N-terminus of theTM, more preferably towards to C-terminus of the TM.

In one embodiment, the non-cytotoxic protease component forms adisulphide bond with the TM component of the fusion protein. In thisregard, the amino acid residue of the protease component that forms thedisulphide bond is preferably located within the last 20, morepreferably within the last 10 C-terminal amino acid residues of theprotease component. Similarly, the amino acid residue within the TMcomponent that forms the second part of the disulphide bond ispreferably located within the last 20, more preferably within the last10 C-terminal amino acid residues of the TM.

The above disulphide bond arrangements have the advantage that theprotease and translocation components are arranged in a manner similarto that for native clostridial neurotoxin. By way of comparison,referring to the primary amino acid sequence for native clostridialneurotoxin, the respective cysteine amino acid residues are distancedapart by between 8 and 27 amino acid residues—taken from Popoff, M R &Marvaud, J-C, 1999, Structural & genomic features of clostridialneurotoxins, Chapter 9, in The Comprehensive Sourcebook of BacterialProtein Toxins. Ed. Alouf & Freer:

‘Native’ length Serotype¹ Sequence between C-C BoNT/A1CVRGIITSKTKS----LDKGYNKALNDLC 23 BoNT/A2 CVRGIIPFKTKS----LDEGYNKALNDLC23 BoNT/B CKSVKAPG-------------------IC 8 BoNT/CCHKAIDGRS----------LYNKTLDC 15 BoNT/D CLRLTK---------------NSRDDSTC 12BoNT/E CKN-IVSVK----------GIRK---SIC 13 BoNT/FCKS-VIPRK----------GTKAPP-RLC 15 BoNT/G CKPVMYKNT----------GKSE----QC 13TeNT CKKIIPPTNIRENLYNRTASLTDLGGELC 27 ¹Information from proteolyticstrains only

The fusion protein may comprise one or more purification tags, which arelocated N-terminal to the protease component and/or C-terminal to thetranslocation component.

Whilst any purification tag may be employed, the following arepreferred:

His-tag (e.g. 6× histidine), preferably as a C-terminal and/orN-terminal tagMBP-tag (maltose binding protein), preferably as an N-terminal tagGST-tag (glutathione-S-transferase), preferably as an N-terminal tagHis-MBP-tag, preferably as an N-terminal tagGST-MBP-tag, preferably as an N-terminal tagThioredoxin-tag, preferably as an N-terminal tagCBD-tag (Chitin Binding Domain), preferably as an N-terminal tag.

According to a further embodiment of the present invention, one or morepeptide spacer molecules may be included in the fusion protein. Forexample, a peptide spacer may be employed between a purification tag andthe rest of the fusion protein molecule (e.g. between an N-terminalpurification tag and a protease component of the present invention;and/or between a C-terminal purification tag and a translocationcomponent of the present invention). A peptide spacer may be alsoemployed between the TM and translocation components of the presentinvention.

A variety of different spacer molecules may be employed in any of thefusion proteins of the present invention. Examples of such spacermolecules include those illustrated in FIGS. 28 and 29. Particularmention here is made to GS15, GS20, GS25, and Hx27—see FIGS. 28 and 29.

The present inventors have unexpectedly found that the fusion proteins(eg. CPNv/A) of the present invention may demonstrate an improvedbinding activity for nociceptive sensory afferents when the size of thespacer is selected so that (in use) the C-terminus of the TM and theN-terminus of the translocation component are separated from one anotherby 40-105 angstroms, preferably by 50-100 angstroms, and more preferablyby 50-90 angstroms. In another embodiment, the preferred spacers have anamino acid sequence of 11-29 amino acid residues, preferably 15-27 aminoacid residues, and more preferably 20-27 amino acid residues. Suitablespacers may be routinely identified and obtained according to Crasto, C.J. and Feng, J. A. (2000) May, 13(5), pp. 309-312—see alsohttp://www.fccc./edu/research/labs/feng/limker.html.

In accordance with a second aspect of the present invention, there isprovided a DNA sequence that encodes the above-mentioned single chainpolypeptide. In a preferred aspect of the present invention, the DNAsequence is prepared as part of a DNA vector, wherein the vectorcomprises a promoter and terminator.

In a preferred embodiment, the vector has a promoter selected from:

Promoter Induction Agent Typical Induction Condition Tac (hybrid) IPTG0.2 mM (0.05-2.0 mM) AraBAD L-arabinose 0.2% (0.002-0.4%) T7-lacoperator IPTG 0.2 mM (0.05-2.0 mM)

The DNA construct of the present invention is preferably designed insilico, and then synthesised by conventional DNA synthesis techniques.

The above-mentioned DNA sequence information is optionally modified forcodon-biasing according to the ultimate host cell (e.g. E. coli)expression system that is to be employed.

The DNA backbone is preferably screened for any inherent nucleic acidsequence, which when transcribed and translated would produce an aminoacid sequence corresponding to the protease cleave site encoded by thesecond peptide-coding sequence. This screening may be performed manuallyor with the assistance of computer software (e.g. the MapDraw program byDNASTAR, Inc.).

According to a further embodiment of the present invention, there isprovided a method of preparing a non-cytotoxic agent, comprising:

-   -   a. contacting a single-chain polypeptide fusion protein of the        invention with a protease capable of cleaving the protease        cleavage site;    -   b. cleaving the protease cleavage site, and thereby forming a        di-chain fusion protein.

This aspect provides a di-chain polypeptide, which generally mimics thestructure of clostridial holotoxin. In more detail, the resultingdi-chain polypeptide typically has a structure wherein:

-   -   a. the first chain comprises the non-cytotoxic protease, or a        fragment thereof, which protease or protease fragment is capable        of cleaving a protein of the exocytic fusion apparatus of a        nociceptive sensory afferent;    -   b. the second chain comprises the TM and the translocation        domain that is capable of translocating the protease or protease        fragment from within an endosome, across the endosomal membrane        and into the cytosol of the nociceptive sensory afferent; and    -   the first and second chains are disulphide linked together.

In use, the single chain or di-chain polypeptide of the invention treat,prevent or ameliorate pain.

In use, a therapeutically effective amount of a single chain or di-chainpolypeptide of the invention is administered to a patient.

The present invention addresses a wide range of pain conditions, inparticular chronic pain conditions. Preferred conditions includecancerous and non-cancerous pain, inflammatory pain and neuropathicpain. The opioid-fusions of the present application are particularlysuited to addressing inflammatory pain, though may be less suited toaddressing neuropathic pain. The galanin-fusions are more suited toaddressing neuropathic pain.

In use, the polypeptides of the present invention are typically employedin the form of a pharmaceutical composition in association with apharmaceutical carrier, diluent and/or excipient, although the exactform of the composition may be tailored to the mode of administration.Administration is preferably to a mammal, more preferably to a human.

The polypeptides may, for example, be employed in the form of a sterilesolution for intra-articular administration or intra-cranialadministration. Spinal injection (e.g. epidural or intrathecal) ispreferred.

The dosage ranges for administration of the polypeptides of the presentinvention are those to produce the desired therapeutic effect. It willbe appreciated that the dosage range required depends on the precisenature of the components, the route of administration, the nature of theformulation, the age of the patient, the nature, extent or severity ofthe patient's condition, contraindications, if any, and the judgement ofthe attending physician.

Suitable daily dosages are in the range 0.0001-1 mg/kg, preferably0.0001-0.5 mg/kg, more preferably 0.002-0.5 mg/kg, and particularlypreferably 0.004-0.5 mg/kg. The unit dosage can vary from less that 1microgram to 30 mg, but typically will be in the region of 0.01 to 1 mgper dose, which may be administered daily or preferably less frequently,such as weekly or six monthly.

A particularly preferred dosing regimen is based on 2.5 ng of fusionprotein (e.g. CPNv/A) as the 1× dose. In this regard, preferred dosagesare in the range 1-−100× (i.e. 2.5-250 ng). This dosage range issignificantly lower (i.e. at least 10-fold, typically 100-fold lower)than would be employed with other types of analgesic molecules such asNSAIDS, morphine, and gabapentin. Moreover, the above-mentioneddifference is considerably magnified when the same comparison is made ona molar basis—this is because the fusion proteins of the presentinvention have a considerably greater Mw than do conventional ‘small’molecule therapeutics.

Wide variations in the required dosage, however, are to be expecteddepending on the precise nature of the components, and the differingefficiencies of various routes of administration.

Variations in these dosage levels can be adjusted using standardempirical routines for optimisation, as is well understood in the art.

Compositions suitable for injection may be in the form of solutions,suspensions or emulsions, or dry powders which are dissolved orsuspended in a suitable vehicle prior to use.

Fluid unit dosage forms are typically prepared utilising a pyrogen-freesterile vehicle. The active ingredients, depending on the vehicle andconcentration used, can be either dissolved or suspended in the vehicle.

In preparing administrable solutions, the polypeptides can be dissolvedin a vehicle, the solution being made isotonic if necessary by additionof sodium chloride and sterilised by filtration through a sterile filterusing aseptic techniques before filling into suitable sterile vials orampoules and sealing. Alternatively, if solution stability is adequate,the solution in its sealed containers may be sterilised by autoclaving.

Advantageously additives such as buffering, solubilising, stabilising,preservative or bactericidal, suspending or emulsifying agents may bedissolved in the vehicle.

Dry powders which are dissolved or suspended in a suitable vehicle priorto use may be prepared by filling pre-sterilised drug substance andother ingredients into a sterile container using aseptic technique in asterile area.

Alternatively the polypeptides and other ingredients may be dissolved inan aqueous vehicle, the solution is sterilized by filtration anddistributed into suitable containers using aseptic technique in asterile area. The product is then freeze dried and the containers aresealed aseptically.

Parenteral suspensions, suitable for intramuscular, subcutaneous orintradermal injection, are prepared in substantially the same manner,except that the sterile components are suspended in the sterile vehicle,instead of being dissolved and sterilisation cannot be accomplished byfiltration. The components may be isolated in a sterile state oralternatively it may be sterilised after isolation, e.g. by gammairradiation.

Advantageously, a suspending agent for example polyvinylpyrrolidone isincluded in the composition's to facilitate uniform distribution of thecomponents.

DEFINITIONS SECTION

Targeting Moiety (TM) means any chemical structure associated with anagent that functionally interacts with a Binding Site to cause aphysical association between the agent and the surface of a target cell.In the context of the present invention, the target cell is anociceptive sensory afferent. The term TM embraces any molecule (i.e. anaturally occurring molecule, or a chemically/physically modifiedvariant thereof) that is capable of binding to a Binding Site on thetarget cell, which Binding Site is capable of internalisation (e.g.endosome formation)—also referred to as receptor-mediated endocytosis.The TM may possess an endosomal membrane translocation function, inwhich case separate TM and Translocation Domain components need not bepresent in an agent of the present invention.

The TM of the present invention binds (preferably specifically binds) toa nociceptive sensory afferent (e.g. a primary nociceptive afferent). Inthis regard, specifically binds means that the TM binds to a nociceptivesensory afferent (e.g. a primary nociceptive afferent) with a greateraffinity than it binds to other neurons such as non-nociceptiveafferents, and/or to motor neurons (i.e. the natural target forclostridial neurotoxin holotoxin). The term “specifically binding” canalso mean that a given TM binds to a given receptor, for example theORL₁ receptor, with a binding affinity (Ka) of 10⁶ M⁻¹ or greater,preferably 10⁷ M⁻¹ or greater, more preferably 10⁸ M⁻¹ or greater, andmost preferably, 10⁹ M⁻¹ or greater.

For the purposes of this invention, an agonist is defined as a moleculethat is capable of stimulating the process of exocytic fusion in atarget cell, which process is susceptible to inhibition by a protease(or fragment thereof) capable of cleaving a protein of the exocyticfusion apparatus in said target cell.

Accordingly, the particular agonist definition of the present inventionwould exclude many molecules that would be conventionally considered asagonists.

For example, nerve growth factor (NGF) is an agonist in respect of itsability to promote neuronal differentiation via binding to a TrkAreceptor. However, NGF is not an agonist when assessed by the abovecriteria because it is not a principal inducer of exocytic fusion. Inaddition, the process that NGF stimulates (i.e. cell differentiation) isnot susceptible to inhibition by the protease activity of anon-cytotoxic toxin molecule.

The term “fragment”, when used in relation to a protein, means a peptidehaving at least thirty-five, preferably at least twenty-five, morepreferably at least twenty, and most preferably at least ten amino acidresidues of the protein in question.

The term “variant”, when used in relation to a protein, means a peptideor peptide fragment of the protein that contains one or more analoguesof an amino acid (e.g. an unnatural amino acid), or a substitutedlinkage.

The term “derivative”, when used in relation to a protein, means aprotein that comprises the protein in question, and a further peptidesequence. The further peptide sequence should preferably not interferewith the basic folding and thus conformational structure of the originalprotein. Two or more peptides (or fragments, or variants) may be joinedtogether to form a derivative. Alternatively, a peptide (or fragment, orvariant) may be joined to an unrelated molecule (e.g. a second,unrelated peptide). Derivatives may be chemically synthesized, but willbe typically prepared by recombinant nucleic acid methods. Additionalcomponents such as lipid, and/or polysaccharide, and/or polyketidecomponents may be included.

Throughout this specification, reference to the “ORL₁ receptor” embracesall members of the ORL₁ receptor family. Members of the ORL₁ receptorfamily typically have a seven transmembrane domain structure and arecoupled to G-proteins of the G_(i) and G₀ families. A method fordetermining the G-protein-stimulating activity of ligands of the ORL₁receptor is given in Example 12. A method for measuring reduction incellular cAMP levels following ORL₁ activation is given in Example 11. Afurther characteristic of members of the ORL₁ receptor family is thatthey are typically able to bind nociceptin (the natural ligand of ORL₁).As an example, all alternative splice variants of the ORL₁ receptor, aremembers of the ORL₁ receptor family.

The term non-cytotoxic means that the protease molecule in question doesnot kill the target cell to which it has been re-targeted.

The protease of the present invention embraces all naturally-occurringnon-cytotoxic proteases that are capable of cleaving one or moreproteins of the exocytic fusion apparatus in eukaryotic cells.

The protease of the present invention is preferably a bacterial protease(or fragment thereof). More preferably the bacterial protease isselected from the genera Clostridium or Neisseria (e.g. a clostridialL-chain, or a neisserial IgA protease preferably from N. gonorrhoeae).

The present invention also embraces modified non-cytotoxic proteases,which include amino acid sequences that do not occur in nature and/orsynthetic amino acid residues, so long as the modified proteases stilldemonstrate the above-mentioned protease activity.

The protease of the present invention preferably demonstrates a serineor metalloprotease activity (e.g. endopeptidase activity). The proteaseis preferably specific for a SNARE protein (e.g. SNAP-25,synaptobrevin/VAMP, or syntaxin).

Particular mention is made to the protease domains of neurotoxins, forexample the protease domains of bacterial neurotoxins. Thus, the presentinvention embraces the use of neurotoxin domains, which occur in nature,as well as recombinantly prepared versions of said naturally-occurringneurotoxins.

Exemplary neurotoxins are produced by clostridia, and the termclostridial neurotoxin embraces neurotoxins produced by C. tetani(TeNT), and by C. botulinum (BoNT) serotypes A-G, as well as the closelyrelated BoNT-like neurotoxins produced by C. baratii and C. butyricum.The above-mentioned abbreviations are used throughout the presentspecification. For example, the nomenclature BoNT/A denotes the sourceof neurotoxin as BoNT (serotype A). Corresponding nomenclature appliesto other BoNT serotypes.

The term L-chain fragment means a component of the L-chain of aneurotoxin, which fragment demonstrates a metalloprotease activity andis capable of proteolytically cleaving a vesicle and/or plasma membraneassociated protein involved in cellular exocytosis.

A Translocation Domain is a molecule that enables translocation of aprotease (or fragment thereof) into a target cell such that a functionalexpression of protease activity occurs within the cytosol of the targetcell. Whether any molecule (e.g. a protein or peptide) possesses therequisite translocation function of the present invention may beconfirmed by any one of a number of conventional assays.

For example, Shone C. (1987) describes an in vitro assay employingliposomes, which are challenged with a test molecule. Presence of therequisite translocation function is confirmed by release from theliposomes of K⁺ and/or labelled NAD, which may be readily monitored [seeShone C. (1987) Eur. J. Biochem; vol. 167(1): pp. 175-180].

A further example is provided by Blaustein R. (1987), which describes asimple in vitro assay employing planar phospholipid bilayer membranes.The membranes are challenged with a test molecule and the requisitetranslocation function is confirmed by an increase in conductance acrosssaid membranes [see Blaustein (1987) FEBS Letts; vol. 226, no. 1: pp.115-120].

Additional methodology to enable assessment of membrane fusion and thusidentification of Translocation Domains suitable for use in the presentinvention are provided by Methods in Enzymology Vol 220 and 221,Membrane Fusion Techniques, Parts A and B, Academic Press 1993.

The Translocation Domain is preferably capable of formation ofion-permeable pores in lipid membranes under conditions of low pH.Preferably it has been found to use only those portions of the proteinmolecule capable of pore-formation within the endosomal membrane.

The Translocation Domain may be obtained from a microbial proteinsource, in particular from a bacterial or viral protein source. Hence,in one embodiment, the Translocation Domain is a translocating domain ofan enzyme, such as a bacterial toxin or viral protein.

It is well documented that certain domains of bacterial toxin moleculesare capable of forming such pores. It is also known that certaintranslocation domains of virally expressed membrane fusion proteins arecapable of forming such pores. Such domains may be employed in thepresent invention.

The Translocation Domain may be of a clostridial origin, namely theH_(N) domain (or a functional component thereof). H_(N) means a portionor fragment of the H-chain of a clostridial neurotoxin approximatelyequivalent to the amino-terminal half of the H-chain, or the domaincorresponding to that fragment in the intact H-chain. It is preferredthat the H-chain substantially lacks the natural binding function of theH_(C) component of the H-chain. In this regard, the H_(C) function maybe removed by deletion of the H_(C) amino acid sequence (either at theDNA synthesis level, or at the post-synthesis level by nuclease orprotease treatment). Alternatively, the H_(C) function may beinactivated by chemical or biological treatment. Thus, the H-chain ispreferably incapable of binding to the Binding Site on a target cell towhich native clostridial neurotoxin (i.e. holotoxin) binds.

In one embodiment, the translocation domain is a H_(N) domain (or afragment thereof) of a clostridial neurotoxin. Examples of suitableclostridial Translocation Domains include:

-   -   Botulinum type A neurotoxin-amino acid residues (449-871)    -   Botulinum type B neurotoxin-amino acid residues (441-858)    -   Botulinum type C neurotoxin-amino acid residues (442-866)    -   Botulinum type D neurotoxin-amino acid residues (446-862)    -   Botulinum type E neurotoxin-amino acid residues (423-845)    -   Botulinum type F neurotoxin-amino acid residues (440-864)    -   Botulinum type G neurotoxin-amino acid residues (442-863)    -   Tetanus neurotoxin-amino acid residues (458-879)

For further details on the genetic basis of toxin production inClostridium botulinum and C. tetani, we refer to Henderson et al (1997)in The Clostridia: Molecular Biology and Pathogenesis, Academic press.

The term H_(N) embraces naturally-occurring neurotoxin H_(N) portions,and modified H_(N) portions having amino acid sequences that do notoccur in nature and/or synthetic amino acid residues, so long as themodified H_(N) portions still demonstrate the above-mentionedtranslocation function.

Alternatively, the Translocation Domain may be of a non-clostridialorigin (see Table 4). Examples of non-clostridial Translocation Domainorigins include, but not be restricted to, the translocation domain ofdiphtheria toxin [O=Keefe et al., Proc. Natl. Acad. Sci. USA (1992) 89,6202-6206; Silverman et al., J. Biol. Chem. (1993) 269, 22524-22532; andLondon, E. (1992) Biochem. Biophys. Acta., 1112, pp. 25-51], thetranslocation domain of Pseudomonas exotoxin type A [Prior et al.Biochemistry (1992) 31, 3555-3559], the translocation domains of anthraxtoxin [Blanke et al. Proc. Natl. Acad. Sci. USA (1996) 93, 8437-8442], avariety of fusogenic or hydrophobic peptides of translocating function[Plank et al. J. Biol. Chem. (1994) 269, 12918-12924; and Wagner et al(1992) PNAS, 89, pp. 7934-7938], and amphiphilic peptides [Murata et al(1992) Biochem., 31, pp. 1986-1992]. The Translocation Domain may mirrorthe Translocation Domain present in a naturally-occurring protein, ormay include amino acid variations so long as the variations do notdestroy the translocating ability of the Translocation Domain.

Particular examples of viral Translocation Domains suitable for use inthe present invention include certain translocating domains of virallyexpressed membrane fusion proteins. For example, Wagner et al. (1992)and Murata et al. (1992) describe the translocation (i.e. membranefusion and vesiculation) function of a number of fusogenic andamphiphilic peptides derived from the N-terminal region of influenzavirus haemagglutinin. Other virally expressed membrane fusion proteinsknown to have the desired translocating activity are a translocatingdomain of a fusogenic peptide of Semliki Forest Virus (SFV), atranslocating domain of vesicular stomatitis virus (VSV) glycoprotein G,a translocating domain of SER virus F protein and a translocating domainof Foamy virus envelope glycoprotein. Virally encoded Aspike proteinshave particular application in the context of the present invention, forexample, the E1 protein of SFV and the G protein of the G protein ofVSV.

Use of the Translocation Domains listed in Table (below) includes use ofsequence variants thereof. A variant may comprise one or moreconservative nucleic acid substitutions and/or nucleic acid deletions orinsertions, with the proviso that the variant possesses the requisitetranslocating function. A variant may also comprise one or more aminoacid substitutions and/or amino acid deletions or insertions, so long asthe variant possesses the requisite translocating function.

Translocation Amino acid domain source residues References Diphtheriatoxin 194-380 Silverman et al., 1994, J. Biol. Chem. 269, 22524-22532London E., 1992, Biochem. Biophys. Acta., 1113, 25-51 Domain II of405-613 Prior et al., 1992, pseudomonas Biochemistry 31, exotoxin3555-3559 Kihara & Pastan, 1994, Bioconj Chem. 5, 532-538 Influenzavirus GLFGAIAGFIENGWE Plank et al., 1994, haemagglutinin GMIDGWYG, andJ. Biol. Chem. 269, Variants thereof 12918-12924 Wagner et al., 1992,PNAS, 89, 7934-7938 Murata et al., 1992, Biochemistry 31, 1986-1992Semliki Forest virus Translocation domain Kielian et al., 1996,fusogenic protein J Cell Biol. 134(4), 863-872 Vesicular Stomatitis118-139 Yao et al., 2003, virus glycoprotein G Virology 310(2), 319-332SER virus F protein Translocation domain Seth et al., 2003, J Virol77(11) 6520-6527 Foamy virus Translocation domain Picard-Maureau et al.,envelope 2003, J Virol. 77(8), glycoprotein 4722-4730

FIGURES

FIG. 1 Purification of a LC/A-nociceptin-H_(N)/A fusion protein

FIG. 2 Purification of a nociceptin-LC/A-H_(N)/A fusion protein

FIG. 3 Purification of a LC/C-nociceptin-H_(N)/C fusion protein

FIG. 4 Purification of a LC/A-met enkephalin-H_(N)/A fusion protein

FIG. 5 Comparison of binding efficacy of a LC/A-nociceptin-H_(N)/Afusion protein and a nociceptin-LC/A-H_(N)/A fusion protein

FIG. 6 In vitro catalytic activity of a LC/A-nociceptin-H_(N)/A fusionprotein

FIG. 7 Purification of a LC/A-nociceptin variant-H_(N)/A fusion protein

FIG. 8 Comparison of binding efficacy of a LC/A-nociceptin-H_(N)/Afusion protein and a LC/A-nociceptin variant-H_(N)/A fusion protein

FIG. 9 Expressed/purified LC/A-nociceptin-H_(N)/A fusion protein familywith variable spacer length product(s)

FIG. 10 Inhibition of SP release and cleavage of SNAP-25 by CPN-A

FIG. 11 Inhibition of SP release and cleavage of SNAP-25 over extendedtime periods after exposure of DRG to CPN-A

FIG. 12 Cleavage of SNAP-25 by CPNv-A

FIG. 13 Cleavage of SNAP-25 over extended time periods after exposure ofDRG to CPNv-A

FIG. 14 CPNv-A fusion-mediated displacement of [3H]-nociceptin binding

FIG. 15 Expressed/purified CPNv(Ek)-A product

FIG. 16 Cleavage of SNAP-25 by CPNv(Ek)-A

FIG. 17 Expressed/purified CPNv-C product

FIG. 18 Cleavage of syntaxin by CPNv-C

FIG. 19 CPN-A efficacy in the Acute Capsaicin-Induced MechanicalAllodynia model

FIG. 20 CPN-A efficacy in the Streptozotocin (STZ)-Induced PeripheralDiabetic Neuropathy (Neuropathic Pain) model

FIG. 21 CPNv-A efficacy in the Acute Capsaicin-Induced MechanicalAllodynia model

FIG. 22 Expressed/purified LC/A-CPLE-H_(N)/A product

FIG. 23 Expressed/purified LC/A-CPBE-H_(N)/A product

FIG. 24 Expressed/purified CPOP-A product

FIG. 25 Expressed/purified CPOPv-A product

FIG. 26 In vitro SNAP-25 cleavage in a DRG cell model

FIG. 27 Expressed/purified CPNv-A-FXa-HT (removable his-tag)

FIG. 28 In vitro efficacy of LC/A-nociceptin-H_(N)/A fusion proteinswith variable spacer length, as assessed by ligand competition assay

FIG. 29 In vitro efficacy of LC/A-nociceptin-H_(N)/A fusion proteinswith variable spacer length, as assessed by in vitro SNAP-25 cleavage

The Figures are now described in more detail.

FIG. 1—Purification of a LC/A-nociceptin-H_(N)/A Fusion Protein

Using the methodology outlined in Example 9, a LC/A-nociceptin-H_(N)/Afusion protein was purified from E. coli BL21 cells. Briefly, thesoluble products obtained following cell disruption were applied to anickel-charged affinity capture column. Bound proteins were eluted with100 mM imidazole, treated with Factor Xa to activate the fusion proteinand remove the maltose-binding protein (MBP) tag, then re-applied to asecond nickel-charged affinity capture column. Samples from thepurification procedure were assessed by SDS-PAGE (Panel A) and Westernblotting (Panel B). Anti-nociceptin antisera (obtained from Abcam) wereused as the primary antibody for Western blotting. The final purifiedmaterial in the absence and presence of reducing agent is identified inthe lanes marked [−] and [+] respectively.

FIG. 2—Purification of a nociceptin-LC/A-H_(N)/A Fusion Protein

Using the methodology outlined in Example 9, a nociceptin-LC/A-H_(N)/Afusion protein was purified from E. coli BL21 cells. Briefly, thesoluble products obtained following cell disruption were applied to anickel-charged affinity capture column. Bound proteins were eluted with100 mM imidazole, treated with Factor Xa to activate the fusion proteinand remove the maltose-binding protein (MBP) tag, then re-applied to asecond nickel-charged affinity capture column. Samples from thepurification procedure were assessed by SDS-PAGE (Panel A) and Westernblotting (Panel B). Anti-nociceptin antisera (obtained from Abcam) wereused as the primary antibody for Western blotting. The final purifiedmaterial in the absence and presence of reducing agent is identified inthe lanes marked [−] and [+] respectively.

FIG. 3—Purification of a LC/C-nociceptin-H_(N)/C Fusion Protein

Using the methodology outlined in Example 9, an LC/C-nociceptin-H_(N)/Cfusion protein was purified from E. coli BL21 cells. Briefly, thesoluble products obtained following cell disruption were applied to anickel-charged affinity capture column. Bound proteins were eluted with100 mM imidazole, treated with Factor Xa to activate the fusion proteinand remove the maltose-binding protein (MBP) tag, then re-applied to asecond nickel-charged affinity capture column. Samples from thepurification procedure were assessed by SDS-PAGE (Panel A) and Westernblotting (Panel B). Anti-nociceptin antisera (obtained from Abcam) wereused as the primary antibody for Western blotting. The final purifiedmaterial in the absence and presence of reducing agent is identified inthe lanes marked [−] and [+] respectively.

FIG. 4—Purification of a LC/A-met enkephalin-H_(N)/A Fusion Protein

Using the methodology outlined in Example 9, an LC/A-metenkephalin-H_(N)/A fusion protein was purified from E. coli BL21 cells.Briefly, the soluble products obtained following cell disruption wereapplied to a nickel-charged affinity capture column. Bound proteins wereeluted with 100 mM imidazole, treated with Factor Xa to activate thefusion protein and remove the maltose-binding protein (MBP) tag, thenre-applied to a second nickel-charged affinity capture column. Samplesfrom the purification procedure were assessed by SDS-PAGE. The finalpurified material in the absence and presence of reducing agent isidentified in the lanes marked [−] and [+] respectively.

FIG. 5—Comparison of Binding Efficacy of a LC/A-nociceptin-H_(N)/AFusion Protein and a nociceptin-LC/A-H_(N)/A Fusion Protein

The ability of nociceptin fusions to bind to the ORL₁ receptor wasassessed using a simple competition-based assay. Primary cultures ofdorsal root ganglia (DRG) were exposed to varying concentrations of testmaterial in the presence of 1 nM [3H]-nociceptin. The reduction inspecific binding of the radiolabelled ligand was assessed byscintillation counting, and plotted in comparison to the efficacy ofunlabelled ligand (Tocris nociceptin). It is clear that theLC/A-nociceptin-H_(N)/A fusion is far superior to thenociceptin-LC/A-H_(N)/A fusion at interacting with the ORL₁ receptor.

FIG. 6—In Vitro Catalytic Activity of a LC/A-nociceptin-H_(N)/A FusionProtein

The in vitro endopeptidase activity of the purifiedLC/A-nociceptin-H_(N)/A fusion protein was determined essentially asdescribed in Chaddock et al 2002, Prot. Express Purif. 25, 219-228.Briefly, SNAP-25 peptide immobilised to an ELISA plate was exposed tovarying concentrations of fusion protein for 1 hour at 37° C. Followinga series of washes, the amount of cleaved SNAP-25 peptide was quantifiedby reactivity with a specific antisera.

FIG. 7—Purification of a LC/A-nociceptin variant-H_(N)/A Fusion Protein

Using the methodology outlined in Example 9, an LC/A-nociceptinvariant-H_(N)/A fusion protein was purified from E. coli BL21 cells.Briefly, the soluble products obtained following cell disruption wereapplied to a nickel-charged affinity capture column. Bound proteins wereeluted with 100 mM imidazole, treated with Factor Xa to activate thefusion protein and remove the maltose-binding protein (MBP) tag, thenre-applied to a second nickel-charged affinity capture column. Samplesfrom the purification procedure were assessed by SDS-PAGE. The finalpurified material in the absence and presence of reducing agent isidentified in the lanes marked [−] and [+] respectively.

FIG. 8—Comparison of Binding Efficacy of a LC/A-nociceptin-H_(N)/AFusion Protein and a LC/A-nociceptin variant-H_(N)/A Fusion Protein

The ability of nociceptin fusions to bind to the ORL₁ receptor wasassessed using a simple competition-based assay. Primary cultures ofdorsal root ganglia (DRG) were exposed to varying concentrations of testmaterial in the presence of 1 nM [3H]-nociceptin. The reduction inspecific binding of the radiolabelled ligand was assessed byscintillation counting, and plotted in comparison to the efficacy ofunlabelled ligand (Tocris nociceptin). It is clear that theLC/A-nociceptin variant-H_(N)/A fusion (CPNv-LHA) is superior to theLC/A-nociceptin variant-H_(N)/A fusion (CPN-LHA) at interacting with theORL₁ receptor.

FIG. 9—Expressed/Purified LC/A-nociceptin-H_(N)/A Fusion Protein Familywith Variable Spacer Length Product(S)

Using the methodology outlined in Example 9, variants of theLC/A-CPN-H_(N)/A fusion consisting of GS10, GS30 and HX27 are purifiedfrom E. coli cell paste. Samples from the purification ofLC/A-CPN(GS10)-H_(N)/A, LC/A-CPN(GS15)-H_(N)/A, LC/A-CPN(GS25)-H_(N)/A,LC/A-CPN(GS30)-H_(N)/A and LC/A-CPN(HX27)-H_(N)/A were assessed bySDS-PAGE prior to staining with Coomassie Blue. The electrophoresisprofile indicates purification of a disulphide-bonded di-chain speciesof the expected molecular mass of CPBE-A. Top panel: M=benchmarkmolecular mass markers; S=total E. coli protein soluble fraction;FT=proteins that did not bind to the Ni²⁺-charged Sepharose column;FUSION=fusion protein eluted by the addition of imidazole. Bottom panel:Lane 1=benchmark molecular mass markers; Lane 2=total E. coli proteinsoluble fraction; Lane 3=purified material following initial capture onNi²⁺-charged Sepharose; Lane 4=Factor Xa treated material prior to finalcapture on Ni²⁺-charged Sepharose; Lane 5=purified final material postactivation with Factor Xa (5 μl); Lane 6=purified final material postactivation with Factor Xa (10 μl); Lane 7=purified final material postactivation with Factor Xa (20 μl); Lane 8=purified final material postactivation with Factor Xa+DTT (5 μl); Lane 9=purified final materialpost activation with Factor Xa+DTT (10 μl); Lane 10=purified finalmaterial post activation with Factor Xa+DTT (20 μl).

FIG. 10—Inhibition of SP Release and Cleavage of SNAP-25 by CPN-A

Briefly, primary cultures of dorsal root ganglia (DRG) were exposed tovarying concentrations of CPN-A for 24 hours. Cellular proteins wereseparated by SDS-PAGE, Western blotted, and probed with anti-SNAP-25 tofacilitate an assessment of SNAP-25 cleavage. The percentage of cleavedSNAP-25 was calculated by densitometric analysis and plotted againstfusion concentration (dashed line). Material was also recovered for ananalysis of substance P content using a specific EIA kit. Inhibition ofsubstance P release is illustrated by the solid line. The fusionconcentration required to achieve 50% maximal SNAP-25 cleavage isestimated to be 6.30±2.48 nM.

FIG. 11—Inhibition of SP Release and Cleavage of SNAP-25 Over ExtendedTime Periods after Exposure of DRG to CPN-A

Primary cultures of dorsal root ganglia (DRG) were exposed to varyingconcentrations of CPN-A for 24 hours. Botulinum neurotoxin (BoNT/A) wasused as a control. After this initial exposure, extracellular materialwas removed by washing, and the cells incubated at 37° C. for varyingperiods of time. At specific time points, cellular proteins wereseparated by SDS-PAGE, Western blotted, and probed with anti-SNAP-25 tofacilitate an assessment of SNAP-25 cleavage. The percentage of cleavedSNAP-25 was calculated by densitometric analysis and illustrated by thedotted lines. Material was also recovered for an analysis of substance Pcontent using a specific EIA kit. Inhibition of substance P release isillustrated by the solid lines.

FIG. 12—Cleavage of SNAP-25 by CPNv-A

Primary cultures of dorsal root ganglia (DRG) were exposed to varyingconcentrations of CPNv-A for 24 hours. Cellular proteins were separatedby SDS-PAGE, Western blotted, and probed with anti-SNAP-25 to facilitatean assessment of SNAP-25 cleavage. The percentage of cleaved SNAP-25 wascalculated by densitometric analysis. The fusion concentration requiredto achieve 50% maximal SNAP-25 cleavage is estimated to be 1.38±0.36 nM.

FIG. 13—Cleavage of SNAP-25 Over Extended Time Periods after Exposure ofDRG to CPNv-A

Primary cultures of dorsal root ganglia (DRG) were exposed to varyingconcentrations of CPNv-A for 24 hours. CPN-A was used as a control.After this initial exposure, extracellular material was removed bywashing, and the cells incubated at 37° C. for varying periods of time.At specific time points, cellular proteins were separated by SDS-PAGE,Western blotted, and probed with anti-SNAP-25 to facilitate anassessment of SNAP-25 cleavage. The percentage of cleaved SNAP-25 wascalculated by densitometric analysis.

FIG. 14—CPNv-A Fusion-Mediated Displacement of [3H]-nociceptin Binding

The ability of nociceptin fusions to bind to the ORL₁ receptor wasassessed using a simple competition-based assay. Primary cultures ofdorsal root ganglia (DRG) were exposed to varying concentrations of testmaterial in the presence of 1 nM [3H]-nociceptin. The reduction inspecific binding of the radiolabelled ligand was assessed byscintillation counting, and plotted in comparison to the efficacy ofunlabelled ligand (Tocris nociceptin). It is clear that theLC/A-nociceptin variant-H_(N)/A fusion (labelled as CPNv-LHnA) issuperior to the LC/A-nociceptin-H_(N)/A fusion (labelled as CPN-LHnA) atinteracting with the ORL₁ receptor.

FIG. 15—Expressed/Purified CPNv(Ek)-A Product

Proteins were subjected to SDS-PAGE prior to staining with CoomassieBlue. The electrophoresis profile indicates purification of adisulphide-bonded di-chain species of the expected molecular mass ofCPNv(Ek)-A. Lane 1=benchmark molecular mass markers; Lane 2=total E.coli protein soluble fraction; Lane 3=purified material followinginitial capture on Ni²⁺-charged Sepharose; Lane 4=purified finalmaterial post activation with enterokinase (5 μl); Lane 5=purified finalmaterial post activation with enterokinase (10 μl); Lane 6=purifiedfinal material post activation with enterokinase (20 μl); Lane7=purified final material post activation with enterokinase+DTT (5 μl);Lane 8=purified final material post activation with enterokinase+DTT (10μl); Lane 9=purified final material post activation withenterokinase+DTT (20 μl).

FIG. 16—Cleavage of SNAP-25 by CPNv(Ek)-A

Primary cultures of dorsal root ganglia (DRG) were exposed to varyingconcentrations of CPNv(Ek)-A for 24 hours. Cellular proteins wereseparated by SDS-PAGE, Western blotted, and probed with anti-SNAP-25 tofacilitate an assessment of SNAP-25 cleavage. The percentage of cleavedSNAP-25 was calculated by densitometric analysis. CPNv-A as prepared inExample 9 was used for comparison purposes. The percentage cleavage ofSNAP-25 by CPNv(Ek)-A (labelled as En activated) and CPNv-A (labelled asXa activated) are illustrated.

FIG. 17—Expressed/Purified CPNv-C Product

Proteins were subjected to SDS-PAGE prior to staining with CoomassieBlue. The electrophoresis profile indicates purification of adisulphide-bonded di-chain species of the expected molecular mass ofCPNv-C. Lane 1=benchmark molecular mass markers; Lane 2=total E. coliprotein soluble fraction; Lane 3=purified material following initialcapture on Ni²⁺-charged Sepharose; Lane 4=Factor Xa treated materialprior to final capture on Ni²⁺-charged Sepharose; Lane 5=purifiedmaterial following second capture on Ni²⁺-charged Sepharose; Lane6=final purified material; Lane 7=final purified material+DTT; Lane8=benchmark molecular mass markers.

FIG. 18—Cleavage of Syntaxin by CPNv-C

Primary cultures of dorsal root ganglia (DRG) were exposed to varyingconcentrations of CPNv-C for 24 hours. Cellular proteins were separatedby SDS-PAGE, Western blotted, and probed with anti-syntaxin tofacilitate an assessment of syntaxin cleavage. The percentage of cleavedsyntaxin was calculated by densitometric analysis. The fusionconcentration required to achieve 50% maximal syntaxin cleavage isestimated to be 3.13±1.96 nM.

FIG. 19—CPN-A Efficacy in the Acute Capsaicin-Induced MechanicalAllodynia Model

The ability of an LC/A-nociceptin-H_(N)/A fusion (CPN/A) to inhibitcapsaicin-induced mechanical allodynia was evaluated followingsubcutaneous intraplantar injection in the rat hind paw. Test animalswere evaluated for paw withdrawal frequency (PWF %) in response to a 10g Von Frey filament stimulus series (10 stimuli×3 trials) prior torecruitment into the study (Pre-Treat); after subcutaneous intraplantartreatment with CPN/A but before capsaicin (Pre-CAP); and followingcapsaicin challenge post-injection of CPN/A (average of responses at 15′and 30′; CAP). Capsaicin challenge was achieved by injection of 10 μL ofa 0.3% solution. Sample dilutions were prepared in 0.5% BSA/saline.

FIG. 20—CPN-A Efficacy in the Streptozotocin (STZ)-Induced PeripheralDiabetic Neuropathy (Neuropathic Pain) Model

Male Sprague-Dawley rats (250-300 g) are treated with 65 mg/kg STZ incitrate buffer (I.V.) and blood glucose and lipid are measured weekly todefine the readiness of the model. Paw Withdrawal Threshold (PWT) ismeasured in response to a Von Frey filament stimulus series over aperiod of time. Allodynia is said to be established when the PWT on twoconsecutive test dates (separated by 1 week) measures below 6 g on thescale. At this point, rats are randomized to either a saline group(negative efficacy control), gabapentin group (positive efficacycontrol) or a test group (CPN/A). Test materials (20-25 μl) are injectedsubcutaneously as a single injection (except gabapentin) and the PWT ismeasured at 1 day post-treatment and periodically thereafter over a 2week period. Gabapentin (30 mg/kg i.p. @ 3 ml/kg injection volume) isinjected daily, 2 hours prior to the start of PWT testing.

FIG. 21—CPNv-A Efficacy in the Acute Capsaicin-Induced MechanicalAllodynia Model

The ability of an LC/A-nociceptin variant-H_(N)/A fusion (CPNv/A) toinhibit capsaicin-induced mechanical allodynia was evaluated followingsubcutaneous intraplantar injection in the rat hind paw. Test animalswere evaluated for paw withdrawal frequency (PWF %) in response to a 10g Von Frey filament stimulus series (10 stimuli×3 trials) prior torecruitment into the study (Pre-Treat), after subcutaneous intraplantartreatment with CPNv/A but before capsaicin (Pre-CAP), and followingcapsaicin challenge post-injection of CPNv/A (average of responses at15′ and 30′; CAP). Capsaicin challenge was achieved by injection of 10μL of a 0.3% solution. Sample dilutions were prepared in 0.5%BSA/saline. These data are expressed as a normalized paw withdrawalfrequency differential, in which the difference between the peakresponse (post-capsaicin) and the baseline response (pre-capsaicin) isexpressed as a percentage. With this analysis, it can be seen thatCPNv/A is more potent than CPN/A since a lower dose of CPNv/A isrequired to achieve similar analgesic effect to that seen with CPN/A.

FIG. 22—Expressed/Purified LC/A-CPLE-H_(N)/A Product

Proteins were subjected to SDS-PAGE prior to staining with CoomassieBlue. The electrophoresis profile indicates purification of adisulphide-bonded di-chain species of the expected molecular mass ofCPLE-A. Lane 1=benchmark molecular mass markers; Lane 2=total E. coliprotein soluble fraction; Lane 3=purified material following initialcapture on Ni²⁺-charged Sepharose; Lane 4=Factor Xa treated materialprior to final capture on Ni²⁺-charged Sepharose; Lane 5=purifiedmaterial following second capture on Ni²⁺-charged Sepharose; Lane6=final purified material; Lane 7=final purified material+DTT.

FIG. 23—Expressed/Purified LC/A-CPBE-H_(N)/A Product

Proteins were subjected to SDS-PAGE prior to staining with CoomassieBlue. The electrophoresis profile indicates purification of adisulphide-bonded di-chain species of the expected molecular mass ofCPBE-A. Lane 1=total E. coli protein soluble fraction; Lane 2=purifiedmaterial following initial capture on Ni²⁺-charged Sepharose; Lane3=Factor Xa treated material prior to final capture on Ni²⁺-chargedSepharose; Lane 4=purified final material post activation with Factor Xa(5 μl); Lane 5=purified final material post activation with Factor Xa(10 μl); Lane 6=purified final material post activation with Factor Xa(20 μl); Lane 7=purified final material post activation with FactorXa+DTT (5 μl); Lane 8=purified final material post activation withFactor Xa+DTT (10 μl); Lane 9=purified final material post activationwith Factor Xa+DTT (20 μl); Lane 10=benchmark molecular mass markers.

FIG. 24—Expressed/Purified CPOP-A Product

Proteins were subjected to SDS-PAGE prior to staining with CoomassieBlue. The electrophoresis profile indicates purification of adisulphide-bonded di-chain species of the expected molecular mass ofCPOP-A. Lane 1=benchmark molecular mass markers; Lane 2=purifiedmaterial following initial capture on Ni²⁺-charged Sepharose; Lane3=Factor Xa treated material prior to final capture on Ni²⁺-chargedSepharose; Lane 4=purified material following second capture onNi²⁺-charged Sepharose; Lane 5=purified final material post activationwith Factor Xa (5 μl); Lane 6=purified final material post activationwith Factor Xa (10 μl); Lane 7=purified final material post activationwith Factor Xa (20 μl); Lane 8=purified final material post activationwith Factor Xa+DTT (5 μl); Lane 9=purified final material postactivation with Factor Xa+DTT (10 μl); Lane 10=purified final materialpost activation with Factor Xa+DTT (20 μl).

FIG. 25—Expressed/Purified CPOPv-A Product

Proteins were subjected to SDS-PAGE prior to staining with CoomassieBlue. The electrophoresis profile indicates purification of adisulphide-bonded di-chain species of the expected molecular mass ofCPOPv-A. Lane 1=benchmark molecular mass markers; Lane 2=total E. coliprotein soluble fraction; Lane 3=purified material following initialcapture on Ni²⁺-charged Sepharose; Lane 4=Factor Xa treated materialprior to final capture on Ni²⁺-charged Sepharose; Lane 5=purified finalmaterial post activation with Factor Xa (5 μl); Lane 6=purified finalmaterial post activation with Factor Xa (10 μl); Lane 7=purified finalmaterial post activation with Factor Xa (20 μl); Lane 8=purified finalmaterial post activation with Factor Xa+DTT (5 μl); Lane 9=purifiedfinal material post activation with Factor Xa+DTT (10 μl); Lane10=purified final material post activation with Factor Xa+DTT (20 μl).

FIG. 26—In Vitro SNAP-25 Cleavage in a DRG Cell Model

Primary cultures of dorsal root ganglia (DRG) were exposed to varyingconcentrations of CPOPv-A for 24 hours. Cellular proteins were separatedby SDS-PAGE, Western blotted, and probed with anti-SNAP-25 to facilitatean assessment of SNAP-25 cleavage. The percentage of cleaved SNAP-25 wascalculated by densitometric analysis.

FIG. 27—Expressed/Purified CPNv-A-FXa-HT (Removable his-tag)

Proteins were subjected to SDS-PAGE prior to staining with CoomassieBlue. The electrophoresis profile indicates purification of adisulphide-bonded di-chain species of the expected molecular mass ofCPNv-A-FXa-HT. Lane 1=benchmark molecular mass markers; Lane 2=total E.coli protein soluble fraction; Lane 3=Factor Xa treated material priorto final capture on Ni²⁺-charged Sepharose; Lane 4=purified finalmaterial post activation with Factor Xa; Lane 5=purified final materialpost activation with Factor Xa+DTT.

FIG. 28—In Vitro Efficacy of LC/A-nociceptin-H_(N)/A Fusion Proteinswith Variable Spacer Length, as Assessed by Ligand Competition Assay

The ability of LC/A-nociceptin-H_(N)/A fusions of variable spacer lengthto bind to the ORL₁ receptor was assessed using a simplecompetition-based assay. Primary cultures of dorsal root ganglia (DRG)were exposed to varying concentrations of test material in the presenceof 1 nM [3H]-nociceptin. The reduction in specific binding of theradiolabelled ligand was assessed by scintillation counting, and plottedin comparison to the efficacy of unlabelled ligand (Tocris nociceptin).The upper panel illustrates the displacement characteristics of the GS0,GS20, GS30 and Hx27 spacers, whilst the lower panel illustrates thedisplacement achieved by the GS10, GS15 and GS25 spaced fusion proteins.It is concluded that the GS0 and GS30 spacers are ineffective, and theGS10 is poorly effective, at displacing nociceptin from the ORL1receptor.

FIG. 29—In Vitro Efficacy of LC/A-nociceptin-H_(N)/A Fusion Proteinswith Variable Spacer Length, as Assessed by in Vitro SNAP-25 Cleavage

Primary cultures of dorsal root ganglia (DRG) were exposed to varyingconcentrations of CPN-A (of variable spacer length) for 24 hours.Cellular proteins were separated by SDS-PAGE, Western blotted, andprobed with anti-SNAP-25 to facilitate an assessment of SNAP-25cleavage. The percentage of cleaved SNAP-25 was calculated bydensitometric analysis. The poorly effective binding characteristics ofthe GS10 spaced fusion protein (see FIG. 28) are reflected in the higherconcentrations of fusion required to achieve cleavage of intracellularSNAP-25. GS0 and GS30 spaced fusion proteins were completely ineffective(date not shown). GS15, 20 and 25 spaced fusion proteins were similarlyeffective.

SEQ ID NOs

SEQ ID1 DNA sequence of the LC/ASEQ ID2 DNA sequence of the H_(N)/ASEQ ID3 DNA sequence of the LC/BSEQ ID4 DNA sequence of the H_(N)/BSEQ ID5 DNA sequence of the LC/CSEQ ID6 DNA sequence of the H_(N)/CSEQ ID7 DNA sequence of the CPN-A linkerSEQ ID8 DNA sequence of the A linkerSEQ ID9 DNA sequence of the N-terminal presentation nociceptin insertSEQ ID10 DNA sequence of the CPN-C linkerSEQ ID11 DNA sequence of the CPBE-A linkerSEQ ID12 DNA sequence of the CPNvar-A linkerSEQ ID13 DNA sequence of the LC/A-CPN-H_(N)/A fusionSEQ ID14 Protein sequence of the LC/A-CPN-H_(N)/A fusionSEQ ID15 DNA sequence of the N-LC/A-H_(N)/A fusionSEQ ID16 Protein sequence of the N-LC/A-H_(N)/A fusionSEQ ID17 DNA sequence of the LC/C-CPN-H_(N)/C fusionSEQ ID18 Protein sequence of the LC/C-CPN-H_(N)/C fusionSEQ ID19 DNA sequence of the LC/C-CPN-H_(N)/C (A-linker) fusionSEQ ID20 Protein sequence of the LC/C-CPN-H_(N)/C (A-linker) fusionSEQ ID21 DNA sequence of the LC/A-CPME-H_(N)/A fusionSEQ ID22 Protein sequence of the LC/A-CPME-H_(N)/A fusionSEQ ID23 DNA sequence of the LC/A-CPBE-H_(N)/A fusionSEQ ID24 Protein sequence of the LC/A-CPBE-H_(N)/A fusionSEQ ID25 DNA sequence of the LC/A-CPNv-H_(N)/A fusionSEQ ID26 Protein sequence of the LC/A-CPNv-H_(N)/A fusionSEQ ID27 DNA sequence of the LC/A-CPN[1-11]-HN/A fusionSEQ ID28 Protein sequence of the LC/A-CPN[1-11]-HN/A fusionSEQ ID29 DNA sequence of the LC/A-CPN[[Y10]1-11]-HN/A fusionSEQ ID30 Protein sequence of the LC/A-CPN[[Y10]1-11]-HN/A fusionSEQ ID31 DNA sequence of the LC/A-CPN[[Y11]1-11]-HN/A fusionSEQ ID32 Protein sequence of the LC/A-CPN[[Y11]1-11]-HN/A fusionSEQ ID33 DNA sequence of the LC/A-CPN[[Y14]1-17]-HN/A fusionSEQ ID34 Protein sequence of the LC/A-CPN[[Y14]1-17]-HN/A fusionSEQ ID35 DNA sequence of the LC/A-CPN[1-13]-HN/A fusionSEQ ID36 Protein sequence of the LC/A-CPN[1-13]-HN/A fusionSEQ ID37 DNA sequence of CPN[1-17]

SEQ ID38 Protein Sequence of CPN[1-17]

SEQ ID39 DNA sequence of CPN[1-11]SEQ ID40 Protein sequence of CPN[1-11]SEQ ID41 DNA sequence of CPN[[Y10]1-11]SEQ ID42 Protein sequence of CPN[[Y10]1-11]SEQ ID43 DNA sequence of CPN[[Y11]1-11]SEQ ID44 Protein sequence of CPN[[Y11]1-11]SEQ ID45 DNA sequence of CPN[[Y14]1-17]SEQ ID46 Protein sequence of CPN[[Y14]1-17]SEQ ID47 DNA sequence of CPN[1-13]SEQ ID48 Protein sequence of CPN[1-13]SEQ ID49 DNA sequence of CPNv (also known as N[[R14K15]1-17])SEQ ID50 Protein sequence of CPNv (also known as N[[R14K15]1-17])SEQ ID51 DNA sequence of the nociceptin-spacer-LC/A-H_(N)/A fusionSEQ ID52 Protein sequence of the nociceptin-spacer-LC/A-H_(N)/A fusionSEQ ID53 DNA sequence of the CPN-A GS10 linkerSEQ ID54 DNA sequence of the CPN-A GS15 linkerSEQ ID55 DNA sequence of the CPN-A GS25 linkerSEQ ID56 DNA sequence of the CPN-A GS30 linkerSEQ ID57 DNA sequence of the CPN-A HX27 linkerSEQ ID58 DNA sequence of the LC/A-CPN(GS15)-H_(N)/A fusionSEQ ID59 Protein sequence of the LC/A-CPN(GS15)-H_(N)/A fusionSEQ ID60 DNA sequence of the LC/A-CPN(GS25)-H_(N)/A fusionSEQ ID61 Protein sequence of the LC/A-CPN(GS25)-H_(N)/A fusionSEQ ID62 DNA sequence of the CPNvar-A Enterokinase activatable linkerSEQ ID63 DNA sequence of the LC/A-CPNv(Ek)-H_(N)/A fusionSEQ ID64 Protein sequence of the LC/A-CPNv(Ek)-H_(N)/A fusionSEQ ID65 DNA sequence of the CPNvar-A linkerSEQ ID66 DNA sequence of the LC/C-CPNv-H_(N)/C fusion (act. A)SEQ ID67 Protein sequence of the LC/C-CPNv-H_(N)/C fusion (act. A)SEQ ID68 DNA sequence of the LC/A-CPLE-H_(N)/A fusionSEQ ID69 Protein sequence of the LC/A-CPLE-H_(N)/A fusionSEQ ID70 DNA sequence of the LC/A-CPOP-H_(N)/A fusionSEQ ID71 Protein sequence of the LC/A-CPOP-H_(N)/A fusionSEQ ID72 DNA sequence of the LC/A-CPOPv-H_(N)/A fusionSEQ ID73 Protein sequence of the LC/A-CPOPv-H_(N)/A fusionSEQ ID74 DNA sequence of the IgA proteaseSEQ ID75 DNA sequence of the IgA-CPNv-H_(N)/A fusionSEQ ID76 Protein sequence of the IgA-CPNv-H_(N)/A fusionSEQ ID77 DNA sequence of the FXa-HTSEQ ID78 DNA sequence of the CPNv-A-FXa-HTSEQ ID79 Protein sequence of the CPNv-A-FXa-HT fusionSEQ ID80 DNA sequence of the DT translocation domainSEQ ID81 DNA sequence of the CPLE-DT-ASEQ ID82 Protein sequence of the CPLE-DT-A fusionSEQ ID83 DNA sequence of the TeNT LCSEQ ID84 DNA sequence of the CPNv-TENT LCSEQ ID85 Protein sequence of the CPNV-TeNT LC fusionSEQ ID86 DNA sequence of the CPNvar-C linkerSEQ ID87 DNA sequence of the LC/C-CPNv-H_(N)/C fusion (act. C)SEQ ID88 Protein sequence of the LC/C-CPNv-H_(N)/C fusion (act. C)

EXAMPLES Example 1 Preparation of a LC/A and H_(N)/A Backbone Clones

The following procedure creates the LC and H_(N) fragments for use asthe component backbone for multidomain fusion expression. This exampleis based on preparation of a serotype A based clone (SEQ ID1 and SEQID2), though the procedures and methods are equally applicable to theother serotypes [illustrated by the sequence listing for serotype B (SEQID3 and SEQ ID4) and serotype C (SEQ ID5 and SEQ ID6)].

Preparation of Cloning and Expression Vectors

pCR 4 (Invitrogen) is the chosen standard cloning vector, selected dueto the lack of restriction sequences within the vector and adjacentsequencing primer sites for easy construct confirmation. The expressionvector is based on the pMAL (NEB) expression vector, which has thedesired restriction sequences within the multiple cloning site in thecorrect orientation for construct insertion (BamHI-SalI-PstI-HindIII). Afragment of the expression vector has been removed to create anon-mobilisable plasmid and a variety of different fusion tags have beeninserted to increase purification options.

Preparation of Protease (e.g. LC/A) Insert

The LC/A (SEQ ID1) is created by one of two ways:

The DNA sequence is designed by back translation of the LC/A amino acidsequence [obtained from freely available database sources such asGenBank (accession number P10845) or Swissprot (accession locusBXA1_CLOBO) using one of a variety of reverse translation software tools(for example EditSeq best E. coli reverse translation (DNASTAR Inc.), orBacktranslation tool v2.0 (Entelechon)]. BamHI/SalI recognitionsequences are incorporated at the 5′ and 3′ ends respectively of thesequence, maintaining the correct reading frame. The DNA sequence isscreened (using software such as MapDraw, DNASTAR Inc.) for restrictionenzyme cleavage sequences incorporated during the back translation. Anycleavage sequences that are found to be common to those required by thecloning system are removed manually from the proposed coding sequenceensuring common E. coli codon usage is maintained. E. coli codon usageis assessed by reference to software programs such as Graphical CodonUsage Analyser (Geneart), and the % GC content and codon usage ratioassessed by reference to published codon usage tables (for exampleGenBank Release 143, 13 Sep. 2004). This optimised DNA sequencecontaining the LC/A open reading frame (ORF) is then commerciallysynthesized (for example by Entelechon, Geneart or Sigma-Genosys) and isprovided in the pCR 4 vector.

The alternative method is to use PCR amplification from an existing DNAsequence with BamHI and SalI restriction enzyme sequences incorporatedinto the 5′ and 3′ PCR primers respectively. Complementaryoligonucleotide primers are chemically synthesised by a supplier (forexample MWG or Sigma-Genosys), so that each pair has the ability tohybridize to the opposite strands (3′ ends pointing “towards” eachother) flanking the stretch of Clostridium target DNA, oneoligonucleotide for each of the two DNA strands. To generate a PCRproduct the pair of short oligonucleotide primers specific for theClostridium DNA sequence are mixed with the Clostridium DNA template andother reaction components and placed in a machine (the ‘PCR machine’)that can change the incubation temperature of the reaction tubeautomatically, cycling between approximately 94° C. (for denaturation),55° C. (for oligonucleotide annealing), and 72° C. (for synthesis).Other reagents required for amplification of a PCR product include a DNApolymerase (such as Taq or Pfu polymerase), each of the four nucleotidedNTP building blocks of DNA in equimolar amounts (50-200 μM) and abuffer appropriate for the enzyme optimised for Mg²⁺ concentration(0.5-5 mM).

The amplification product is cloned into pCR 4 using either, TOPO TAcloning for Taq PCR products or Zero Blunt TOPO cloning for Pfu PCRproducts (both kits commercially available from Invitrogen). Theresultant clone is checked by sequencing. Any additional restrictionsequences which are not compatible with the cloning system are thenremoved using site directed mutagenesis [for example, using Quickchange(Stratagene Inc.)].

Preparation of Translocation (e.g. H_(N)) Insert

The H_(N)/A (SEQ ID2) is created by one of two ways:

The DNA sequence is designed by back translation of the H_(N)/A aminoacid sequence [obtained from freely available database sources such asGenBank (accession number P10845) or Swissprot (accession locusBXA1_CLOBO)] using one of a variety of reverse translation softwaretools [for example EditSeq best E. coli reverse translation (DNASTARInc.), or Backtranslation tool v2.0 (Entelechon)]. A PstI restrictionsequence added to the N-terminus and XbaI-stop codon-HindIII to theC-terminus ensuring the correct reading frame is maintained. The DNAsequence is screened (using software such as MapDraw, DNASTAR Inc.) forrestriction enzyme cleavage sequences incorporated during the backtranslation. Any sequences that are found to be common to those requiredby the cloning system are removed manually from the proposed codingsequence ensuring common E. coli codon usage is maintained. E. colicodon usage is assessed by reference to software programs such asGraphical Codon Usage Analyser (Geneart), and the % GC content and codonusage ratio assessed by reference to published codon usage tables (forexample GenBank Release 143, 13 Sep. 2004). This optimised DNA sequenceis then commercially synthesized (for example by Entelechon, Geneart orSigma-Genosys) and is provided in the pCR 4 vector.

The alternative method is to use PCR amplification from an existing DNAsequence with PstI and XbaI-stop codon-HindIII restriction enzymesequences incorporated into the 5′ and 3′ PCR primers respectively. ThePCR amplification is performed as described above. The PCR product isinserted into pCR 4 vector and checked by sequencing. Any additionalrestriction sequences which are not compatible with the cloning systemare then removed using site directed mutagenesis [for example usingQuickchange (Stratagene Inc.)].

Example 2 Preparation of a LC/A-nociceptin-H_(N)/A Fusion Protein(nociceptin is N-terminal of the H_(N)-chain) Preparation ofLinker-Nociceptin-Spacer Insert

The LC-H_(N) linker can be designed from first principle, using theexisting sequence information for the linker as the template. Forexample, the serotype A linker (in this case defined as the inter-domainpolypeptide region that exists between the cysteines of the disulphidebridge between LC and H_(N)) is 23 amino acids long and has the sequenceVRGIITSKTKSLDKGYNKALNDL. Within this sequence, it is understood thatproteolytic activation in nature leads to an H_(N) domain that has anN-terminus of the sequence ALNDL. This sequence information is freelyavailable from available database sources such as GenBank (accessionnumber P10845) or Swissprot (accession locus BXA1_CLOBO). Into thislinker a Factor Xa site, nociceptin and spacer are incorporated; andusing one of a variety of reverse translation software tools [forexample EditSeq best E. coli reverse translation (DNASTAR Inc.), orBacktranslation tool v2.0 (Entelechon)], the DNA sequence encoding thelinker-ligand-spacer region is determined. Restriction sites are thenincorporated into the DNA sequence and can be arranged asBamHI-SalI-linker-proteasesite-nociceptin-NheI-spacer-SpeI-PstI-XbaI-stop codon-HindIII (SEQ ID7).It is important to ensure the correct reading frame is maintained forthe spacer, nociceptin and restriction sequences and that the XbaIsequence is not preceded by the bases, TC, which would result on DAMmethylation. The DNA sequence is screened for restriction sequenceincorporation, and any additional sequences are removed manually fromthe remaining sequence ensuring common E. coli codon usage ismaintained. E. coli codon usage is assessed by reference to softwareprograms such as Graphical Codon Usage Analyser (Geneart), and the % GCcontent and codon usage ratio assessed by reference to published codonusage tables (for example, GenBank Release 143, 13 Sep. 2004). Thisoptimised DNA sequence is then commercially synthesized (for example byEntelechon, Geneart or Sigma-Genosys) and is provided in the pCR 4vector.

Preparation of the LC/A-nociceptin-H_(A)/A Fusion

In order to create the LC-linker-nociceptin-spacer-H_(N) construct (SEQID13), the pCR 4 vector encoding the linker (SEQ ID7) is cleaved withBamHI+SalI restriction enzymes. This cleaved vector then serves as therecipient vector for insertion and ligation of the LC/A DNA (SEQ ID1)cleaved with BamHI+SalI. The resulting plasmid DNA is then cleaved withPstI+XbaI restriction enzymes and serves as the recipient vector for theinsertion and ligation of the H_(N)/A DNA (SEQ ID2) cleaved withPstI+XbaI. The final construct contains theLC-linker-nociceptin-spacer-H_(N) ORF (SEQ ID13) for transfer intoexpression vectors for expression to result in a fusion protein of thesequence illustrated in SEQ ID14.

Example 3 Preparation of a nociceptin-LC/A-H_(N)/A Fusion Protein(nociceptin is N-terminal of the LC-chain)

The LC/A-H_(N)/A backbone is constructed as described in Example 2 usingthe synthesised A serotype linker with the addition of a Factor Xa sitefor activation, arranged as BamHI-SalI-linker-proteasesite-linker-PstI-XbaI-stop codon-HindIII (SEQ 108). The LC/A-H_(N)/Abackbone and the synthesised N-terminal presentation nociceptin insert(SEQ ID9) are cleaved with BamHI+HindIII restriction enzymes, gelpurified and ligated together to create anociceptin-spacer-LC-linker-H_(N). The ORF (SEQ ID15) is then cut outusing restriction enzymes AvaI+XbaI for transfer into expression vectorsfor expression to result in a fusion protein of the sequence illustratedin SEQ ID16.

Example 4 Preparation of a LC/C-nociceptin-H_(N)/C Fusion Protein

Following the methods used in Examples 1 and 2, the LC/C (SEQ ID5) andH_(N)/C (SEQ ID6) are created and inserted into the C serotype linkerarranged as BamHIH-SalI-linker-proteasesite-nociceptin-NheI-spacer-SpeI-PstI-XbaI-stop codon-HindIII (SEQID10). The final construct contains theLC-linker-nociceptin-spacer-H_(N) ORF (SEQ ID17) for expression as aprotein of the sequence illustrated in SEQ ID18.

Example 5 Preparation of a LC/C-nociceptin-H_(N)/C Fusion Protein with aSerotype A Activation Sequence

Following the methods used in Examples 1 and 2, the LC/C (SEQ ID5) andH_(N)/C (SEQ ID6) are created and inserted into the A serotype linkerarranged as BamHI-SalI-linker-proteasesite-nociceptin-NheI-spacer-SpeI-PstI-XbaI-stop codon-HindIII (SEQ ID7).The final construct contains the LC-linker-nociceptin-spacer-H_(N) ORF(SEQ ID19) for expression as a protein of the sequence illustrated inSEQ ID20.

Example 6 Preparation of a LC/A-met Enkephalin-H_(N)/A Fusion Protein

Due to the small, five-amino acid, size of the met-enkephalin ligand theLC/A-met enkephalin-H_(N)/A fusion is created by site directedmutagenesis [for example using Quickchange (Stratagene Inc.)] using theLC/A-nociceptin-H_(N)/A fusion (SEQ ID13) as a template.Oligonucleotides are designed encoding the YGGFM met-enkephalin peptide,ensuring standard E. coli codon usage is maintained and no additionalrestriction sites are incorporated, flanked by sequences complimentaryto the linker region of the LC/A-nociceptin-H_(N)/A fusion (SEQ ID13)either side on the nociceptin section. The SDM product is checked bysequencing and the final construct containing the LC-linker-metenkephalin-spacer-H_(N) ORF (SEQ ID21) for expression as a protein ofthe sequence illustrated in SEQ ID22.

Example 7 Preparation of a LC/A-β Endorphin-H_(N)/A Fusion Protein

Following the methods used in Examples 1 and 2, the LC/A (SEQ ID1) andH_(N)/A (SEQ ID2) are created and inserted into the A serotype βendorphin linker arranged as BamHI-SalI-linker-protease site-βendorphin-NheI-spacer-SpeI-PstI-XbaI-stop codon-HindIII (SEQ ID11). Thefinal construct contains the LC-linker-β endorphin-spacer-H_(N) ORF (SEQID23) for expression as a protein of the sequence illustrated in SEQID24.

Example 8 Preparation of a LC/A-nociceptin Variant-H_(N)/A FusionProtein

Following the methods used in Examples 1 and 2, the LC/A (SEQ ID1) andH_(N)/A (SEQ ID2) are created and inserted into the A serotypenociceptin variant linker arranged as BamHI-SalI-linker-proteasesite-nociceptin variant-NheI-spacer-SpeI-PstI-XbaI-stop codon-HindIII(SEQ ID12). The final construct contains the LC-linker-nociceptinvariant-spacer-H_(N) ORF (SEQ ID25) for expression as a protein of thesequence illustrated in SEQ ID26.

Example 9 Purification Method for LC/A-nociceptin-H_(N)/A Fusion Protein

Defrost falcon tube containing 25 ml 50 mM HEPES pH 7.2, 200 mM NaCl andapproximately 10 g of E. coli BL21 cell paste. Make the thawed cellpaste up to 80 ml with 50 mM HEPES pH 7.2, 200 mM NaCl and sonicate onice 30 seconds on, 30 seconds off for 10 cycles at a power of 22 micronsensuring the sample remains cool. Spin the lysed cells at 18 000 rpm, 4°C. for 30 minutes. Load the supernatant onto a 0.1M NiSO₄ chargedChelating column (20-30 ml column is sufficient) equilibrated with 50 mMHEPES pH 7.2, 200 mM NaCl. Using a step gradient of 10 and 40 mMimidazol, wash away the non-specific bound protein and elute the fusionprotein with 100 mM imidazol. Dialyse the eluted fusion protein against5 L of 50 mM HEPES pH 7.2, 200 mM NaCl at 4° C. overnight and measurethe OD of the dialysed fusion protein. Add 1 unit of factor Xa per 100μg fusion protein and Incubate at 25° C. static overnight. Load onto a0.1M NiSO₄ charged Chelating column (20-30 ml column is sufficient)equilibrated with 50 mM HEPES pH 7.2, 200 mM NaCl. Wash column tobaseline with 50 mM HEPES pH 7.2, 200 mM NaCl. Using a step gradient of10 and 40 mM imidazol, wash away the non-specific bound protein andelute the fusion protein with 100 mM imidazol. Dialyse the eluted fusionprotein against 5 L of 50 mM HEPES pH 7.2, 200 mM NaCl at 4° C.overnight and concentrate the fusion to about 2 mg/ml, aliquot sampleand freeze at −20° C. Test purified protein using OD, BCA, purityanalysis and SNAP-25 assessments.

Example 10 Confirmation of TM Agonist Activity by Measuring Release ofSubstance P from Neuronal Cell Cultures Materials

Substance P EIA is obtained from R&D Systems, UK.

Methods

Primary neuronal cultures of eDRG are established as describedpreviously (Duggan et al., 2002). Substance P release from the culturesis assessed by EIA, essentially as described previously (Duggan et al.,2002). The TM of interest is added to the neuronal cultures (establishedfor at least 2 weeks prior to treatment); control cultures are performedin parallel by addition of vehicle in place of TM. Stimulated (100 mMKCl) and basal release, together with total cell lysate content, ofsubstance P are obtained for both control and TM treated cultures.Substance P immunoreactivity is measured using Substance P EnzymeImmunoassay Kits (Cayman Chemical Company, USA or R&D Systems, UK)according to manufacturers' instructions.

The amount of Substance P released by the neuronal cells in the presenceof the TM of interest is compared to the release obtained in thepresence and absence of 100 mM KCl. Stimulation of Substance P releaseby the TM of interest above the basal release, establishes that the TMof interest is an “agonist ligand” as defined in this specification. Ifdesired the stimulation of Substance P release by the TM of interest canbe compared to a standard Substance P release-curve produced using thenatural ORL-1 receptor ligand, nociceptin (Tocris).

Example 11 Confirmation of ORL₁ Receptor Activation by MeasuringForskolin-Stimulated cAMP Production

Confirmation that a given TM is acting via the ORL₁ receptor is providedby the following test, in which the TMs ability to inhibitforskolin-stimulated cAMP production is assessed.

Materials

[³H]adenine and [¹⁴C]cAMP are obtained from GE Healthcare

Methods

The test is conducted essentially as described previously by Meunier etal. [Isolation and structure of the endogenous agonist of opioidreceptor-like ORL₁ receptor. Nature 377: 532-535, 1995] in intacttransfected-CHO cells plated on 24-well plastic plates.

To the cells is added [3H]adenine (1.0 μCi) in 0.4 ml of culture medium.The cells remain at 37° C. for 2 h to allow the adenine to incorporateinto the intracellular ATP pool. After 2 h, the cells are washed oncewith incubation buffer containing: 130 mM NaCl, 4.8 mM KCl, 1.2 mMKH₂PO₄, 1.3 mM CaCl₂, 1.2 mM MgSO₄, 10 mM glucose, 1 mg/ml bovine serumalbumin and 25 mM HEPES pH 7.4, and replaced with buffer containingforskolin (10 μM) and isobutylmethylxanthine (50 μM) with or without theTM of interest. After 10 min, the medium is aspirated and replaced with0.5 ml, 0.2 M HCl. Approximately 1000 cpm of [¹⁴C]cAMP is added to eachwell and used as an internal standard. The contents of the wells arethen transferred to columns of 0.65 g dry alumina powder. The columnsare eluted with 4 ml of 5 mM HCl, 0.5 ml of 0.1M ammonium acetate, thentwo additional millilitres of ammonium acetate. The final eluate iscollected into scintillation vials and counted for ¹⁴C and tritium.Amounts collected are corrected for recovery of [¹⁴C]cAMP. TMs that areagonists at the ORL₁ receptor cause a reduction in the level of cAMPproduced in response to forskolin.

Example 12 Confirmation of ORL₁ Receptor Activation Using a GTPγSBinding Functional Assay

Confirmation that a given TM is acting via the ORL₁ receptor is alsoprovided by the following test, a GTPγS binding functional assay.

Materials

[³⁵S]GTPγS is obtained from GE HealthcareWheatgerm agglutinin-coated (SPA) beads are obtained from GE Healthcare

Methods

This assay is carried out essentially as described by Traynor andNahorski [Modulation by μ-opioid agonists of guanosine-5—O-(3-[³⁵S]thio)triphosphate binding to membranes from humanneuroblastoma SH-SY5Y cells. Mol. Pharmacol. 47: 848-854, 1995].

Cells are scraped from tissue culture dishes into 20 mM HEPES, 1 mMethylenediaminetetraacetic acid, then centrifuged at 500×g for 10 min.Cells are resuspended in this buffer and homogenized with a PolytronHomogenizer.

The homogenate is centrifuged at 27,000×g for 15 min, and the pelletresuspended in buffer A, containing: 20 mM HEPES, 10 mM MgCl₂, 100 mMNaCl, pH 7.4. The suspension is recentrifuged at 20,000×g and suspendedonce more in buffer A. For the binding assay, membranes (8-15 μgprotein) are incubated with [³⁵S]GTP S (50 μM), GDP (10 μM), with andwithout the TM of interest, in a total volume of 1.0 ml, for 60 min at25° C. Samples are filtered over glass fibre filters and counted asdescribed for the binding assays.

Example 13 Preparation of a LC/A-nociceptin-H_(N)/A Fusion Protein(nociceptin is N-terminal of the H_(N)-chain)

The linker-nociceptin-spacer insert is prepared as described in Example2.

Preparation of the LC/A-nociceptin-H_(N)/A Fusion

In order to create the LC-linker-nociceptin-spacer-H_(N) construct (SEQID13), the pCR 4 vector encoding the linker (SEQ ID7) is cleaved withBamHI+SalI restriction enzymes. This cleaved vector then serves as therecipient for insertion and ligation of the LC/A DNA (SEQ ID1) alsocleaved with BamHI+SalI. The resulting plasmid DNA is then cleaved withBamHI+HindIII restriction enzymes and the LC/A-linker fragment insertedinto a similarly cleaved vector containing a unique multiple cloningsite for BamHI, SalI, PstI, and HindIII such as the pMAL vector (NEB).The H_(N)/A DNA (SEQ ID2) is then cleaved with PstI+HindIII restrictionenzymes and inserted into the similarly cleaved pMAL-LC/A-linkerconstruct. The final construct contains theLC-linker-nociceptin-spacer-H_(N) ORF (SEQ ID13) for expression as aprotein of the sequence illustrated in SEQ ID14.

Example 14 Preparation of a nociceptin-LC/A-H_(N)/A Fusion Protein(nociceptin is N-terminal of the LC-chain)

In order to create the nociceptin-spacer-LC/A-H_(N)/A construct, an Aserotype linker with the addition of a Factor Xa site for activation,arranged as BamHI-SalI-linker-protease site-linker-PstI-XbaI-stopcodon-HindIII (SEQ ID8) is synthesised as described in Example 13. ThepCR 4 vector encoding the linker is cleaved with BamHI+SalI restrictionenzymes. This cleaved vector then serves as the recipient for insertionand ligation of the LC/A DNA (SEQ ID1) also cleaved with BamHI+SalI. Theresulting plasmid DNA is then cleaved with BamHI+HindIII restrictionenzymes and the LC/A-linker fragment inserted into a similarly cleavedvector containing the synthesised N-terminal presentation nociceptininsert (SEQ ID9). This construct is then cleaved with AvaI+HindIII andinserted into an expression vector such as the pMAL plasmid (NEB). TheH_(N)/A DNA (SEQ ID2) is then cleaved with PstI+HindIII restrictionenzymes and inserted into the similarly cleavedpMAL-nociceptin-LC/A-linker construct. The final construct contains thenociceptin-spacer-LC/A-H_(N)/A ORF (SEQ ID51) for expression as aprotein of the sequence illustrated in SEQ ID52.

Example 15 Preparation and Purification of an LC/A-nociceptin-H_(N)/AFusion Protein Family with Variable Spacer Length

Using the same strategy as employed in Example 2, a range of DNA linkerswere prepared that encoded nociceptin and variable spacer content. Usingone of a variety of reverse translation software tools [for exampleEditSeq best E. coli reverse translation (DNASTAR Inc.), orBacktranslation tool v2.0 (Entelechon)], the DNA sequence encoding thelinker-ligand-spacer region is determined. Restriction sites are thenincorporated into the DNA sequence and can be arranged asBamHI-SalI-linker-proteasesite-nociceptin-NheI-spacer-SpeI-PstI-XbaI-stop codon-HindIII (SEQ ID53to SEQ ID57). It is important to ensure the correct reading frame ismaintained for the spacer, nociceptin and restriction sequences and thatthe XbaI sequence is not preceded by the bases, TC which would result onDAM methylation. The DNA sequence is screened for restriction sequenceincorporation and any additional sequences are removed manually from theremaining sequence ensuring common E. coli codon usage is maintained. E.coli codon usage is assessed by reference to software programs such asGraphical Codon Usage Analyser (Geneart), and the % GC content and codonusage ratio assessed by reference to published codon usage tables (forexample GenBank Release 143, 13 Sep. 2004). This optimised DNA sequenceis then commercially synthesized (for example by Entelechon, Geneart orSigma-Genosys) and is provided in the pCR 4 vector.

The spacers that were created included:

TABLE 1 SEQ ID of the Code Protein sequence of the linker linker DNAGS10 ALAGGGGSALVLQ 53 GS15 ALAGGGGSGGGGSALVLQ 54 GS25ALAGGGGSGGGGSGGGGSGGGGSALVLQ 55 GS30 ALAGGGGSGGGGSGGGGSGGGGSGGGGSALVLQ56 HX27 ALAAEAAAKEAAAKEAAAKAGGGGSALVLQ 57

By way of example, in order to create the LC/A-CPN(GS15)-H_(N)/A fusionconstruct (SEQ ID58), the pCR 4 vector encoding the linker (SEQ ID54) iscleaved with BamHI+SalI restriction enzymes. This cleaved vector thenserves as the recipient vector for insertion and ligation of the LC/ADNA (SEQ ID1) also cleaved with BamHI+SalI. The resulting plasmid DNA isthen cleaved with BamHI+HindIII restriction enzymes and the LC/A-linkerfragment inserted into a similarly cleaved vector containing a uniquemultiple cloning site for BamHI, SalI, PstI, and HindIII such as thepMAL vector (NEB). The H_(N)/A DNA (SEQ ID2) is then cleaved withPstI+HindIII restriction enzymes and inserted into the similarly cleavedpMAL-LC/A-linker construct. The final construct contains theLC/A-CPN(GS15)-H_(N)/A ORF (SEQ ID58) for expression as a protein of thesequence illustrated in SEQ ID59.

As a further example, to create the LC/A-CPN(GS25)-H_(N)/A fusionconstruct (SEQ ID60), the pCR 4 vector encoding the linker (SEQ ID55) iscleaved with BamHI+SalI restriction enzymes. This cleaved vector thenserves as the recipient vector for insertion and ligation of the LC/ADNA (SEQ ID1) cleaved with BamHI+SalI. The resulting plasmid DNA is thencleaved with BamHI+HindIII restriction enzymes and the LC/A-linkerfragment inserted into a similarly cleaved vector containing a uniquemultiple cloning site for BamHI, SalI, PstI, and HindIII such as thepMAL vector (NEB). The H_(N)/A DNA (SEQ ID2) is then cleaved withPstI+HindIII restriction enzymes and inserted into the similarly cleavedpMAL-LC/A-linker construct. The final construct contains theLC/A-CPN(GS25)-H_(N)/A ORF (SEQ ID60) for expression as a protein of thesequence illustrated in SEQ ID61.

Variants of the LC/A-CPN-H_(N)/A fusion consisting of GS10, GS30 andHX27 are similarly created. Using the purification methodology describedin Example 9, fusion protein is purified from E. coli cell paste. FIG. 9illustrates the purified product obtained in the case ofLC/A-CPN(GS10)-H_(N)/A, LC/A-CPN(GS15)-H_(N)/A, LC/A-CPN(GS25)-H_(N)/A,LC/A-CPN(GS30)-H_(N)/A and LC/A-CPN(HX27)-H_(N)/A.

Example 16 Assessment of In Vitro Efficacy of an LC/A-nociceptin-H_(N)/AFusion

Fusion protein prepared according to Examples 2 and 9 was assessed inthe eDRG neuronal cell model.

Assays for the inhibition of substance P release and cleavage of SNAP-25have been previously reported (Duggan et al., 2002, J. Biol. Chem., 277,34846-34852). Briefly, dorsal root ganglia neurons are harvested from15-day-old fetal Sprague-Dawley rats and dissociated cells plated onto24-well plates coated with Matrigel at a density of 1×10⁶ cells/well.One day post-plating the cells are treated with 10 μM cytosineβ-D-arabinofuranoside for 48 h. Cells are maintained in Dulbecco'sminimal essential medium supplemented with 5% heat-inactivated fetalbovine serum, 5 mM L-glutamine, 0.6% D-glucose, 2% B27 supplement, and100 ng/ml 2.5 S mouse nerve growth factor. Cultures are maintained for 2weeks at 37° C. in 95% air/5% CO₂ before addition of test materials.

Release of substance P from eDRG is assessed by enzyme-linkedimmunosorbent assay. Briefly, eDRG cells are washed twice with lowpotassium-balanced salt solution (BSS: 5 mM KCl, 137 mM NaCl, 1.2 mMMgCl₂, 5 mM glucose, 0.44 mM KH₂PO₄, 20 mM HEPES, pH 7.4, 2 mM CaCl₂).Basal samples are obtained by incubating each well for 5 min. with 1 mlof low potassium BSS. After removal of this buffer, the cells arestimulated to release by incubation with 1 ml of high potassium buffer(BSS as above with modification to include 100 mM KCl isotonicallybalanced with NaCl) for 5 min. All samples are removed to tubes on iceprior to assay of substance P. Total cell lysates are prepared byaddition of 250 μl of 2M acetic acid/0.1% trifluoroacetic acid to lysethe cells, centrifugal evaporation, and resuspension in 500 μl of assaybuffer. Diluted samples are assessed for substance P content. SubstanceP immunoreactivity is measured using Substance P Enzyme Immunoassay Kits(Cayman Chemical Company or R&D Systems) according to manufacturers'instructions. Substance P is expressed in pg/ml relative to a standardsubstance P curve run in parallel.

SDS-PAGE and Western blot analysis were performed using standardprotocols (Novex). SNAP-25 proteins were resolved on a 12% Tris/glycinepolyacrylamide gel (Novex) and subsequently transferred tonitrocellulose membrane. The membranes were probed with a monoclonalantibody (SMI-81) that recognises cleaved and intact SNAP-25. Specificbinding was visualised using peroxidase-conjugated secondary antibodiesand a chemiluminescent detection system. Cleavage of SNAP-25 wasquantified by scanning densitometry (Molecular Dynamics Personal SI,ImageQuant data analysis software). Percent SNAP-25 cleavage wascalculated according to the formula: (Cleaved SNAP-25/(Cleaved+IntactSNAP-25))×100.

Following exposure of eDRG neurons to an LC/A-nociceptin-H_(N)/A fusion(termed CPN-A), both inhibition of substance P release and cleavage ofSNAP-25 are observed (FIG. 10). After 24 h exposure to the fusion, 50%of maximal SNAP-25 cleavage is achieved by a fusion concentration of6.3±2.5 nM.

The effect of the fusion is also assessed at defined time pointsfollowing a 16 h exposure of eDRG to CPN-A. FIG. 11 illustrates theprolonged duration of action of the CPN-A fusion protein, withmeasurable activity still being observed at 28 days post exposure.

Example 17 Assessment of In Vitro Efficacy of an LC/A-nociceptinVariant-H_(N)/A Fusion

Fusion protein prepared according to Examples 8 and 9 was assessed inthe eDRG neuronal cell mode using the method described in Example 16.

Following exposure of eDRG neurons to an LC/A-nociceptin variant-H_(N)/Afusion (termed CPNv-A), both inhibition of substance P release andcleavage of SNAP-25 are observed. After 24 h exposure to the fusion, 50%of maximal SNAP-25 cleavage is achieved by a fusion concentration of1.4±0.4 nM (FIG. 12).

The effect of the fusion is also assessed at defined time pointsfollowing a 16 h exposure of eDRG to CPN-A. FIG. 13 illustrates theprolonged duration of action of the CPN-A fusion protein, withmeasurable activity still being observed at 24 days post exposure.

The binding capability of the CPNv-A fusion protein is also assessed incomparison to the CPN-A fusion. FIG. 14 illustrates the results of acompetition experiment to determine binding efficacy at the ORL-1receptor. CPNv-A is demonstrated to displace [3H]-nociceptin, therebyconfirming that access to the receptor is possible with the ligand inthe central presentation format.

Example 18 Preparation of an LC/A-nociceptin Variant-H_(N)/A FusionProtein That is Activated by Treatment with Enterokinase

Following the methods used in Examples 1 and 2, the LC/A (SEQ ID1) andH_(N)/A (SEQ ID2) are created and inserted into the A serotypenociceptin variant linker arranged as BamHI-SalI-linker-enterokinaseprotease site-nociceptin variant-NheI-spacer-SpeI-PstI-XbaI-stopcodon-HindIII (SEQ ID62). The final construct contains theLC-linker-nociceptin variant-spacer-H_(N) ORF sequences (SEQ ID63) forexpression as a protein of the sequence illustrated in SEQ ID64. Thefusion protein is termed CPNv(Ek)-A. FIG. 15 illustrates thepurification of CPNv(Ek)-A from E. coli following the methods used inExample 9 but using Enterokinase for activation at 0.00064 μg per 100 μgof fusion protein.

Example 19 Assessment of In Vitro Efficacy of a LC/A-nociceptinvariant-H_(N)/A Fusion that has been Activated by Treatment withEnterokinase

The CPNv(Ek)-A prepared in Example 18 is obtained in a purified form andapplied to the eDRG cell model to assess cleavage of SNAP-25 (usingmethodology from Example 16). FIG. 16 illustrates the cleavage ofSNAP-25 following 24 h exposure of eDRG to CPNv(Ek)-A. The efficiency ofcleavage is observed to be similar to that achieved with the FactorXa-cleaved material, as recorded in Example 17.

Example 20 Preparation of an LC/C-nociceptin variant-H_(N)/C FusionProtein with a Factor Xa Activation Linker Derived from Serotype A

Following the methods used in Example 4, the LC/C (SEQ ID5) and H_(N)/C(SEQ ID6) are created and inserted into the A serotype nociceptinvariant linker arranged as BamHI-SalI-linker-nociceptinvariant-NheI-spacer-SpeI-PstI-XbaI-stop codon-HindIII (SEQ ID65). Thefinal construct contains the LC-linker-nociceptin variant-spacer-H_(N)ORF sequences (SEQ ID66) for expression as a protein of the sequenceillustrated in SEQ ID67. The fusion protein is termed CPNv-C (act. A).FIG. 17 illustrates the purification of CPNv-C (act. A) from E. colifollowing the methods used in Example 9.

Example 21 Assessment of In Vitro Efficacy of an LC/C-nociceptinvariant-H_(N)/C Fusion Protein

Following the methods used in Example 9, the CPNv-C (act. A) prepared inExample 20 is obtained in a purified form and applied to the eDRG cellmodel to assess cleavage of SNAP-25 (using methodology from Example 16).After 24 h exposure to the fusion, 50% of maximal syntaxin cleavage isachieved by a fusion concentration of 3.1±2.0 nM. FIG. 18 illustratesthe cleavage of syntaxin following 24 h exposure of eDRG to CPNv-C (act.A).

Example 22 Assessment of In Vivo Efficacy of an LC/A-nociceptin-HN/AFusion

The ability of an LC/A-nociceptin-H_(N)/A fusion (CPN/A) to inhibitacute capsaicin-induced mechanical allodynia is evaluated followingsubcutaneous intraplantar injection in the rat hind paw. Test animalsare evaluated for paw withdrawal frequency (PWF %) in response to a 10 gVon Frey filament stimulus series (10 stimuli×3 trials) prior torecruitment into the study, after subcutaneous treatment with CPN/A butbefore capsaicin, and following capsaicin challenge post-injection ofCPN/A (average of responses at 15′ and 30′). Capsaicin challenge isachieved by injection of 10 μL of a 0.3% solution. Sample dilutions areprepared in 0.5% BSA/saline. FIG. 19 illustrates the reversal ofmechanical allodynia that is achieved by pre-treatment of the animalswith a range of concentrations of LC/A-nociceptin-HN/A fusion.

The ability of an LC/A-nociceptin-HN/A fusion (CPN/A) to inhibitstreptozotocin (STZ)-induced mechanical (tactile) allodynia in rats isevaluated. STZ-induced mechanical allodynia in rats is achieved byinjection of streptozotocin (i.p. or i.v.) which yields destruction ofpancreatic β-cells leading to loss of insulin production, withconcomitant metabolic stress (hyperglycemia and hyperlipidemia). Assuch, STZ induces Type I diabetes. In addition, STZ treatment leads toprogressive development of neuropathy, which serves as a model ofchronic pain with hyperalgesia and allodynia that may reflect signsobserved in diabetic humans (peripheral diabetic neuropathy).

Male Sprague-Dawley rats (250-300 g) are treated with 65 mg/kg STZ incitrate buffer (I.V.) and blood glucose and lipid are measured weekly todefine the readiness of the model. Paw Withdrawal Threshold (PWT) ismeasured in response to a Von Frey filament stimulus series over aperiod of time. Allodynia is said to be established when the PWT on twoconsecutive test dates (separated by 1 week) measures below 6 g on thescale. At this point, rats are randomized to either a saline group(negative efficacy control), gabapentin group (positive efficacycontrol) or a test group (CPN/A). Test materials (20-25 μl) are injectedsubcutaneously as a single injection (except gabapentin) and the PWT ismeasured at 1 day post-treatment and periodically thereafter over a2-week period. Gabapentin (30 mg/kg i.p. @ 3 ml/kg injection volume) isinjected daily, 2 hours prior to the start of PWT testing. FIG. 20illustrates the reversal of allodynia achieved by pre-treatment of theanimals with 750 ng of CPN/A. Data were obtained over a 2-week periodafter a single injection of CPN/A

Example 23 Assessment of In Vivo Efficacy of an LC/A-nociceptinvariant-H_(N)/A Fusion

The ability of an LC/A-nociceptin variant-H_(N)/A fusion (CPNv/A) toinhibit capsaicin-induced mechanical allodynia is evaluated followingsubcutaneous intraplantar injection in the rat hind paw. Test animalsare evaluated for paw withdrawal frequency (PWF %) in response to a 10 gVon Frey filament stimulus series (10 stimuli×3 trials) prior torecruitment into the study (Pre-Treat); after subcutaneous intraplantartreatment with CPNv/A but before capsaicin (Pre-CAP); and followingcapsaicin challenge post-injection of CPNv/A (average of responses at15′ and 30′; CAP). Capsaicin challenge is achieved by injection of 10 μLof a 0.3% solution. Sample dilutions are prepared in 0.5% BSA/saline.

FIG. 21 illustrates the reversal of allodynia that is achieved bypre-treatment of the animals with a range of concentrations ofLC/A-nociceptin variant-H_(N)/A fusion in comparison to the reversalachieved with the addition of LC/A-nociceptin-H_(N)/A fusion. These dataare expressed as a normalized paw withdrawal frequency differential, inwhich the difference between the peak response (post-capsaicin) and thebaseline response (pre-capsaicin) is expressed as a percentage. Withthis analysis, it can be seen that CPNv/A is more potent than CPN/Asince a lower dose of CPNv/A is required to achieve similar analgesiceffect to that seen with CPN/A.

Example 24 Preparation of an LC/A-leu enkephalin-H_(N)/A Fusion Protein

Due to the small, five-amino acid, size of the leu-enkephalin ligand theLC/A-leu enkephalin-H_(N)/A fusion is created by site directedmutagenesis [for example using Quickchange (Stratagene Inc.)] using theLC/A-nociceptin-H_(N)/A fusion (SEQ ID13) as a template.Oligonucleotides are designed encoding the YGGFL leu-enkephalin peptide,ensuring standard E. coli codon usage is maintained and no additionalrestriction sites are incorporated, flanked by sequences complimentaryto the linker region of the LC/A-nociceptin-H_(N)/A fusion (SEQ ID13)either side on the nociceptin section. The SDM product is checked bysequencing and the final construct containing the LC-linker-leuenkephalin-spacer-H_(N) ORF (SEQ ID68) for expression as a protein ofthe sequence illustrated in SEQ ID69. The fusion protein is termedCPLE-A. FIG. 22 illustrates the purification of CPLE-A from E. colifollowing the methods used in Example 9.

Example 25 Expression and Purification of an LC/A-beta-endorphin-H_(N)/AFusion Protein

Following the methods used in Example 9, and with theLC/A-beta-endorphin-H_(N)/A fusion protein (termed CPBE-A) created inExample 7, the CPBE-A is purified from E. coli. FIG. 23 illustrates thepurified protein as analysed by SDS-PAGE.

Example 26 Preparation of an LC/A-nociceptin mutant-H_(N)/A FusionProtein

Due to the single amino acid modification necessary to mutate thenociceptin sequence at position 1 from a Phe to a Tyr, theLC/A-nociceptin mutant-H_(N)/A fusion is created by site directedmutagenesis [for example using Quickchange (Stratagene Inc.)] using theLC/A-nociceptin-H_(N)/A fusion (SEQ ID13) as a template.Oligonucleotides are designed encoding tyrosine at position 1 of thenociceptin sequence, ensuring standard E. coli codon usage is maintainedand no additional restriction sites are incorporated, flanked bysequences complimentary to the linker region of theLC/A-nociceptin-H_(N)/A fusion (SEQ ID13) either side on the nociceptinsection. The SDM product is checked by sequencing and the finalconstruct containing the LC/A-nociceptin mutant-spacer-H_(N)/A fusionORF (SEQ ID70) for expression as a protein of the sequence illustratedin SEQ ID71. The fusion protein is termed CPOP-A. FIG. 24 illustratesthe purification of CPOP-A from E. coli following the methods used inExample 9.

Example 27 Preparation and Assessment of an LC/A-nociceptin Variantmutant-HN/A Fusion Protein

Due to the single amino acid modification necessary to mutate thenociceptin sequence at position 1 from a Phe to a Tyr, theLC/A-nociceptin variant mutant-H_(N)/A fusion is created by sitedirected mutagenesis [for example using Quickchange (Stratagene Inc.)]using the LC/A-nociceptin variant-H_(N)/A fusion (SEQ ID25) as atemplate. Oligonucleotides are designed encoding tyrosine at position 1of the nociceptin sequence, ensuring standard E. coli codon usage ismaintained and no additional restriction sites are incorporated, flankedby sequences complimentary to the linker region of the LC/A-nociceptinvariant-H_(N)/A fusion (SEQ ID25) either side on the nociceptin section.The SDM product is checked by sequencing and the final constructcontaining the LC/A-nociceptin mutant-spacer-H_(N)/A fusion ORF (SEQ072) for expression as a protein of the sequence illustrated in SEQID73. The fusion protein is termed CPOPv-A. FIG. 25 illustrates thepurification of CPOPv-A from E. coli following the methods used inExample 9.

Using methodology described in Example 16, CPOPv-A is assessed for itsability to cleave SNAP-25 in the eDRG cell model. FIG. 26 illustratesthat CPOPv-A is able to cleave SNAP-25 in the eDRG model, achievingcleavage of 50% of the maximal SNAP-25 after exposure of the cells toapproximately 5.9 nM fusion for 24 h.

Example 28 Preparation of an IgA protease-nociceptin variant-H_(N)/AFusion Protein

The IgA protease amino acid sequence was obtained from freely availabledatabase sources such as GenBank (accession number P09790). Informationregarding the structure of the N. Gonorrhoeae IgA protease gene isavailable in the literature (Pohlner et al., Gene structure andextracellular secretion of Neisseria gonorrhoeae IgA protease, Nature,1987, 325(6103), 458-62). Using Backtranslation tool v2.0 (Entelechon),the DNA sequence encoding the IgA protease modified for E. coliexpression was determined. A BamHI recognition sequence was incorporatedat the 5′ end and a codon encoding a cysteine amino acid and SalIrecognition sequence were incorporated at the 3′ end of the IgA DNA. TheDNA sequence was screened using MapDraw, (DNASTAR Inc.) for restrictionenzyme cleavage sequences incorporated during the back translation. Anycleavage sequences that are found to be common to those required forcloning were removed manually from the proposed coding sequence ensuringcommon E. coli codon usage is maintained. E. coli codon usage wasassessed Graphical Codon Usage Analyser (Geneart), and the % GC contentand codon usage ratio assessed by reference to published codon usagetables. This optimised DNA sequence (SEQ ID74) containing the IgA openreading frame (ORF) is then commercially synthesized.

The IgA (SEQ ID74) is inserted into the LC-linker-nociceptinvariant-spacer-H_(N) ORF (SEQ ID25) using BamHI and SalI restrictionenzymes to replace the LC with the IgA protease DNA. The final constructcontains the IgA-linker-nociceptin variant-spacer-H_(N) ORF (SEQ ID75)for expression as a protein of the sequence illustrated in SEQ ID76.

Example 29 Preparation and Assessment of a Nociceptin TargetedEndopeptidase Fusion Protein with a Removable Histidine Purification Tag

DNA was prepared that encoded a Factor Xa removable his-tag (his6),although it is clear that alternative proteases site such asEnterokinase and alternative purification tags such as longer histidinetags are also possible. Using one of a variety of reverse translationsoftware tools [for example EditSeq best E. coli reverse translation(DNASTAR Inc.), or Backtranslation tool v2.0 (Entelechon)], the DNAsequence encoding the Factor Xa removable his-tag region is determined.Restriction sites are then incorporated into the DNA sequence and can bearranged as NheI-linker-SpeI-PstI-H_(N)/A-XbaI-LEIEGRSGHHHHHHStopcodon-HindIII (SEQ ID77). The DNA sequence is screened for restrictionsequence incorporated and any additional sequences are removed manuallyfrom the remaining sequence ensuring common E. coli codon usage ismaintained. E. coli codon usage is assessed by reference to softwareprograms such as Graphical Codon Usage Analyser (Geneart), and the % GCcontent and codon usage ratio assessed by reference to published codonusage tables (for example GenBank Release 143, 13 Sep. 2004). Thisoptimised DNA sequence is then commercially synthesized (for example byEntelechon, Geneart or Sigma-Genosys) and is provided in the pCR 4vector. In order to create CPNv-A-FXa-HT (SEQ ID78, removable his-tagconstruct) the pCR 4 vector encoding the removable his-tag is cleavedwith NheI and HindIII. The NheI−HindIII fragment is then inserted intothe LC/A-CPNv-H_(N)/A vector (SEQ ID25) that has also been cleaved byNheI and HindIII. The final construct contains theLC/A-linker-nociceptin variant-spacer-H_(N)-FXa-Histag-HindIII ORFsequences (SEQ ID78) for expression as a protein of the sequenceillustrated in SEQ ID79. FIG. 27 illustrates the purification ofCPNv-A-FXa-HT from E. coli following the methods used in Example 9.

Example 30 Preparation of a leu-enkephalin Targeted Endopeptidase FusionProtein Containing a Translocation Domain Derived from Diphtheria Toxin

The DNA sequence is designed by back translation of the amino acidsequence of the translocation domain of the diphtheria toxin (obtainedfrom freely available database sources such as GenBank (accession number1×DTT) using one of a variety of reverse translation software tools [forexample EditSeq best E. coli reverse translation (DNASTAR Inc.), orBacktranslation tool v2.0 (Entelechon)]. Restriction sites are thenincorporated into the DNA sequence and can be arranged asNheI-Linker-SpeI-PstI-diphtheria translocation domain-XbaI-stopcodon-HindIII (SEQ ID80). PstI/XbaI recognition sequences areincorporated at the 5′ and 3′ ends of the translocation domainrespectively of the sequence maintaining the correct reading frame. TheDNA sequence is screened (using software such as MapDraw, DNASTAR Inc.)for restriction enzyme cleavage sequences incorporated during the backtranslation. Any cleavage sequences that are found to be common to thoserequired by the cloning system are removed manually from the proposedcoding sequence ensuring common E. coli codon usage is maintained. E.coli codon usage is assessed by reference to software programs such asGraphical Codon Usage Analyser (Geneart), and the % GC content and codonusage ratio assessed by reference to published codon usage tables (forexample GenBank Release 143, 13 Sep. 2004). This optimised DNA sequencecontaining the diphtheria translocation domain is then commerciallysynthesized as NheI-Linker-SpeI-PstI-diphtheria translocationdomain-XbaI-stop codon-HindIII (for example by Entelechon, Geneart orSigma-Genosys) and is provided in the pCR 4 vector (Invitrogen). The pCR4 vector encoding the diphtheria translocation domain is cleaved withNheI and XbaI. The NheI-XbaI fragment is then inserted into theLC/A-CPLE-H_(N)/A vector (SEQ ID68) that has also been cleaved by NheIand XbaI. The final construct contains theLC/A-leu-enkephalin-spacer-diphtheria translocation domain ORF sequences(SEQ ID81) for expression as a protein of the sequence illustrated inSEQ ID82.

Example 31 Preparation of a Nociceptin Variant Targeted EndopeptidaseFusion Protein Containing a LC Domain Derived from Tetanus Toxin

The DNA sequence is designed by back translation of the tetanus toxin LCamino acid sequence (obtained from freely available database sourcessuch as GenBank (accession number X04436) using one of a variety ofreverse translation software tools [for example EditSeq best E. colireverse translation (DNASTAR Inc.), or Backtranslation tool v2.0(Entelechon)]. BamHI/SalI recognition sequences are incorporated at the5′ and 3′ ends respectively of the sequence maintaining the correctreading frame (SEQ ID83). The DNA sequence is screened (using softwaresuch as MapDraw, DNASTAR Inc.) for restriction enzyme cleavage sequencesincorporated during the back translation. Any cleavage sequences thatare found to be common to those required by the cloning system areremoved manually from the proposed coding sequence ensuring common E.coli codon usage is maintained. E. coli codon usage is assessed byreference to software programs such as Graphical Codon Usage Analyser(Geneart), and the % GC content and codon usage ratio assessed byreference to published codon usage tables (for example GenBank Release143, 13 Sep. 2004). This optimised DNA sequence containing the tetanustoxin LC open reading frame (ORF) is then commercially synthesized (forexample by Entelechon, Geneart or Sigma-Genosys) and is provided in thepCR 4 vector (invitrogen). The pCR 4 vector encoding the TeNT LC iscleaved with BamHI and SalI. The BamHI—SalI fragment is then insertedinto the LC/A-CPNv-H_(N)/A vector (SEQ ID25) that has also been cleavedby BamHI and SalI. The final construct contains the TeNTLC-linker-nociceptin variant-spacer-H_(N) ORF sequences (SEQ ID84) forexpression as a protein of the sequence illustrated in SEQ ID85.

Example 32 Preparation of an LC/C-nociceptin variant-H_(N)/C FusionProtein with a Native Serotype C Linker that is Susceptible to Factor XaCleavage

Following the methods used in Example 4, the LC/C (SEQ ID5) and H_(N)/C(SEQ ID6) are created and inserted into the C serotype nociceptinvariant linker arranged as BamHI-SalI-linker-nociceptinvariant-NheI-spacer-SpeI-PstI-XbaI-stop codon-HindIII (SEQ ID86). Thefinal construct contains the LC-linker-nociceptin variant-spacer-H_(N)ORF sequences (SEQ ID87) for expression as a protein of the sequenceillustrated in SEQ ID88. The fusion protein is termed CPNv-C (act. C).

1-29. (canceled)
 30. A method of preventing or suppressing a specificpain condition in a subject, wherein said pain condition is selectedfrom the group consisting of: neuropathic pain, inflammatory pain,headache pain, somatic pain, visceral pain and referred pain, saidmethod comprising administering to said subject a therapeuticallyeffective amount of a polypeptide fusion protein, comprising: a) anon-cytotoxic protease that cleaves a protein of the exocytic fusionapparatus in a nociceptive sensory afferent; b) a Targeting Moiety thatbinds to a Binding Site present on the nociceptive sensory afferent; c)a protease cleavage site located between the non-cytotoxic protease andthe Targeting Moiety; and d) a translocation domain that translocatesthe protease from within an endosome, across an endosomal membrane andinto the cytosol of the nociceptive sensory afferent; wherein theTargeting Moiety is located between the protease cleavage site and thetranslocation domain and wherein, I. the non-cytotoxic protease forms adisulphide bond with the translocation domain and, wherein the aminoacid residue of the non-cytotoxic protease that forms the first part ofthe disulphide bond is located within the last 20 C-terminal amino acidresidues of the non-cytotoxic protease, and the amino acid residuewithin the translocation domain that forms the second part of thedisulphide bond is located within the first 20 N-terminal amino acidresidues of the translocation domain; or II. the non-cytotoxic proteaseforms a disulphide bond with the Targeting Moiety component of thepolypeptide fusion protein, wherein the amino acid residue of thenon-cytotoxic protease that forms the first part of the disulphide bondis located within the last 20 C-terminal amino acid residues of thenon-cytotoxic protease, and the amino acid residue within the TargetingMoiety that forms the second part of the disulphide bond is locatedwithin the last 20 C-terminal amino acid residues of the TargetingMoiety.
 31. The method according to claim 1, wherein the TargetingMoiety and the protease cleavage site are separated by at most 10 aminoacid residues, preferably by at most 5 amino acid residues, and morepreferably by at most zero amino acid residues.
 32. The method accordingto claim 1, wherein the non-cytotoxic protease comprises a clostridianeurotoxin protease or an IgA protease.
 33. The method according toclaim 1, wherein the translocation domain comprises a clostridialneurotoxin peptide.
 34. The method according to claim 1, wherein theTargeting Moiety binds to an ORL1 receptor.
 35. The method according toclaim 1, wherein the Targeting Moiety has at least 80% sequence identityto SEQ ID No. 38 or a fragment thereof.
 36. The method according toclaim 1, wherein the Targeting Moiety has at least 80% sequence identityto SEQ ID No. 40 or a fragment thereof.
 37. The method according toclaim 1, wherein the Targeting Moiety has at least 80% sequence identityto SEQ ID No. 42 or a fragment thereof.
 38. The method according toclaim 1, wherein the Targeting Moiety has at least 80% sequence identityto SEQ ID No. 44 or a fragment thereof.
 39. The method according toclaim 1, wherein the Targeting Moiety has at least 80% sequence identityto SEQ ID No. 46 or a fragment thereof.
 40. The method according toclaim 1, wherein the Targeting Moiety has at least 80% sequence identityto SEQ ID No. 48 or a fragment thereof.
 41. The method according toclaim 1, wherein the Targeting Moiety has at least 80% sequence identityto SEQ ID No. 50 or a fragment thereof.
 42. The method according toclaim 1, wherein the Targeting Moiety is selected from the groupconsisting of: opioid, nociceptin; β-endorphin, endomorphine-1,endomorphine-2, dynorphin, met-enkephalin, leu-enkephalin, galanin, andPAR-2 peptide.
 43. The method according to claim 1, wherein thepolypeptide fusion protein comprises a polypeptide sequence having atleast 80% sequence identity to a polypeptide sequence according to a SEQID NO selected from the group consisting of SEQ ID NOs: 14, 16, 18, 20,22, 24, 26, 28, 30, 32, 34, 36, 52, 59, 61, 64, 67, 69, 71, 73, 76, 79,82, 85, or
 88. 44. A method according to claim 1, wherein thepolypeptide fusion protein is in the form of a di-chain polypeptide thathas been prepared by a process comprising: a) contacting a single-chainpolypeptide fusion protein with a protease capable of cleaving thesingle-chain polypeptide fusion protein; and b) cleaving thesingle-chain polypeptide fusion protein at the protease cleavage site;and thereby forming the di-chain polypeptide.
 45. The method accordingto claim 15, wherein the protease cleavage site is cleaved by a proteaseselected from the group consisting of enterokinase, Factor X, TEV(Tobacco Etch virus), Thrombin, or PreScission.
 46. The method accordingto claim 16, wherein the protease cleavage site is integrated at aposition within the fusion protein such that cleavage of the integratedprotease converts the single-chain fusion protein into the di-chainpolypeptide in which the Targeting Moiety has a free amino terminus thatinteracts directly with the Binding Site.
 47. The method according toclaim 1, wherein the inflammatory pain is selected from a painassociated with an inflammatory condition selected from the groupconsisting of: arthritic disorder; autoimmune disease; connective tissuedisorder; injury; infection; neuritis; or joint inflammation.
 48. Themethod according to claim 1, wherein the headache pain is selected fromthe group consisting of: muscular/myogenic headache; vascular headache;high blood pressure headache; traction and inflammatory headache;hormone headache; rebound headache; chronic sinusitis headache; organicheadache; or ictal headache.
 49. The method according to claim 1,wherein the somatic pain is selected from the group consisting of:excessive muscle tension; repetitive motion disorder; muscle disorder;myalgia; infection; or drugs.
 50. The method according to claim 1,wherein the visceral pain is selected from the group consisting of:functional visceral pain; chronic gastrointestinal inflammation;autoimmune pain; orangic visceral pain; or treatment-induced visceralpain.
 51. The method according to claim 1, wherein the referred pain isselected from the group consisting of pain associated with:intervertebral disc herniation; or myocardial ischaemia.